JP2023513188A - MIRNA-485 inhibitors for increased gene expression - Google Patents

Abstract

The present disclosure provides SIRT1, PGC-1α, CD36, LRRK2, NRG1, STMN2, VLDLR, NRXN1, GRIA4, NXPH1, PSD-95, and/or synaptophysin proteins or SIRT1, PGC-1α, CD36, LRRK2, NRG1, STMN2 , VLDLR, NRXN1, GRIA4, NXPH1, PSD-95, and/or the use of miRNA inhibitors to treat diseases or conditions associated with decreased levels of expression of the synaptophysin genes. In some aspects, miRNA inhibitors can be used to treat diseases or conditions associated with increased levels of caspase-3 protein or gene expression. miRNA inhibitors useful in the present disclosure can inhibit miR-485 expression and/or activity, thereby inhibiting SIRT1, PGC-1α, CD36, LRRK2, NRG1, STMN2, VLDLR, NRXN1, GRIA4, NXPH1 , PSD-95, and/or synaptophysin protein or gene expression can be increased, and/or caspase-3 protein or gene expression can be decreased.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application is filed February 7, 2020, U.S. Provisional Patent Application No. 62/971,767; Claiming the priority benefit of Serial No. 63/047,155 filed July 1, 2020 and Serial No. 63/064,314 filed August 11, 2020; The entirety of each application is incorporated herein by reference.

REFERENCES TO SEQUENCE LISTINGS SUBMITTED ELECTRONICALLY VIA EFS-WEB WITH THIS APPLICATION IN THE FORM OF AN ASCII TEXT FILE (FILE NAME: 4366_014PC04_Seqlisting_ST25.txt, SIZE: 264,023 bytes, CREATED: February 5, 2021) The entire sequence listing submitted electronically is incorporated herein by reference.

The present disclosure includes miR-485 inhibitors (e.g., at least one miR-485 binding site) for treating diseases and disorders associated with decreased SIRT1 expression (e.g., neurodegenerative diseases and disorders, e.g., Alzheimer's disease) Polynucleotide encoding nucleotide molecules) uses.

Sirtuin 1 (also known as NAD-dependent deacetylase sirtuin 1) is an enzyme encoded by the SIRT1 gene in humans. Sirtuin-1 belongs to the family of nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases and can deacetylate a variety of substrates (Rahman, S., et al., Cell Communication and Signaling 9 : 11 (2011)). Sirtuin-1 has therefore been implicated in a wide range of physiological functions, including regulation of gene expression, metabolism, and aging. Moreover, aberrant sirtuin activity has been associated with certain human diseases. For example, subjects with neurodegenerative diseases have been described as having low levels of sirtuin 1 activity.

Neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease are common and increasing causes of mortality and morbidity worldwide. By 2050, it is estimated that more than 100 million people worldwide will develop AD (Gaugler et al., Alzheimer's Dement 12(4):459-509 (2016); Pan et al. ., Sci Adv 5(2) (2019)). The cost of AD is estimated to exceed $800 billion worldwide. Over the past two decades, researchers have attempted to develop compounds and antibodies that can either inhibit the production and accumulation of Aβ or promote the clearance of amyloid beta. Unfortunately, these attempts have not achieved successful clinical utility in large clinical trials with mild AD patients (Panza et al., Nat Rev Neurol 15(2): 73-88 (2019) ).
At present, no therapeutic agent for neurodegenerative diseases is known. The available treatment options are generally limited to palliating various symptoms rather than addressing the underlying causes of the disease. Therefore, new and more effective approaches to the treatment of neurodegenerative diseases are highly desirable.

Rahman, S.; , et al. , Cell Communication and Signaling 9:11 (2011) Gaugler et al. , Alzheimer's Dement 12(4):459-509 (2016); Pan et al. , Sci Adv 5(2) (2019) Panza et al. , Nat Rev Neurol 15(2): 73-88 (2019)

Provided herein is a method for increasing the level of SIRT1 protein and/or SIRT1 gene in a subject in need of increasing the level of SIRT1 protein and/or SIRT1 gene, comprising a compound that inhibits miR-485 (miRNA inhibitor ) to a subject. In some aspects, the subject has a disease or condition associated with decreased levels of SIRT1 protein and/or SIRT1 gene. In some aspects, the miRNA inhibitor induces autophagy and/or treats or prevents inflammation.

Provided herein are methods of increasing CD36 protein and/or CD36 gene levels in a subject in need thereof, comprising compounds that inhibit miR-485 (miRNA inhibitors ) to a subject. In some aspects, the subject has a disease or condition associated with decreased levels of CD36 protein and/or CD36 gene.

The present disclosure further provides a method of increasing levels of PGC-1α protein and/or gene in a subject in need of increasing levels of PGC-1α protein and/or gene, comprising: miR-485 A method comprising administering to a subject a compound that inhibits (miRNA inhibitor) is provided. In some aspects, the subject has a disease or condition associated with decreased levels of PGC-1α protein and/or PGC-1α gene.

Provided herein is a method for increasing the level of LRRK2 protein and/or LRRK2 gene in a subject in need of increasing the level of LRRK2 protein and/or LRRK2 gene, comprising a compound that inhibits miR-485 (miRNA inhibitor ) to a subject. In some aspects, the subject has a disease or condition associated with decreased levels of LRRK2 protein and/or LRRK2 gene.

Provided herein is a method for increasing NRG1 protein and/or NRG1 gene levels in a subject in need of increasing NRG1 protein and/or NRG1 gene levels, comprising a compound that inhibits miR-485 (miRNA inhibitor ) to a subject. In some aspects, the subject has a disease or condition associated with decreased levels of NRG1 protein and/or NRG1 gene.

Provided herein is a method for increasing STMN2 protein and/or STMN2 gene levels in a subject in need of increasing STMN2 protein and/or STMN2 gene levels, comprising a compound that inhibits miR-485 (miRNA inhibitor ) to a subject. In some aspects, the subject has a disease or condition associated with decreased levels of STMN2 protein and/or STMN2 gene.

Provided herein is a method for increasing VLDLR protein and/or VLDLR gene levels in a subject in need of increasing VLDLR protein and/or VLDLR gene levels, comprising a compound that inhibits miR-485 (miRNA inhibitor ) to a subject. In some aspects, the subject has a disease or condition associated with decreased levels of VLDLR protein and/or VLDLR gene.

Provided herein is a method for increasing NRXN1 protein and/or NRXN1 gene levels in a subject in need of increasing NRXN1 protein and/or NRXN1 gene levels, comprising a compound that inhibits miR-485 (miRNA inhibitor ) to a subject. In some aspects, the subject has a disease or condition associated with decreased levels of NRXN1 protein and/or NRXN1 gene.

Provided herein is a method for increasing the level of GRIA4 protein and/or GRIA4 gene in a subject in need of increasing the level of GRIA4 protein and/or GRIA4 gene, comprising a compound that inhibits miR-485 (miRNA inhibitor ) to a subject. In some aspects, the subject has a disease or condition associated with decreased levels of GRIA4 protein and/or GRIA4 gene.

Provided herein is a method for increasing the level of NXPH1 protein and/or gene in a subject in need of increasing the level of NXPH1 protein and/or gene, comprising a compound that inhibits miR-485 (miRNA inhibitor ) to a subject. In some aspects, the subject has a disease or condition associated with decreased levels of NXPH1 protein and/or NXPH1 gene.

Provided herein is a method of increasing PSD-95 protein and/or PSD-95 gene levels in a subject in need thereof, comprising miR-485 A method comprising administering to a subject a compound that inhibits (miRNA inhibitor) is provided. In some aspects, the subject has a disease or condition associated with decreased levels of PSD-95 protein and/or PSD-95 gene.

Provided herein is a method for increasing the level of synaptophysin protein and/or synaptophysin gene in a subject in need thereof, comprising a compound that inhibits miR-485 (miRNA inhibitor ) to a subject. In some aspects, the subject has a disease or condition associated with decreased levels of synaptophysin protein and/or synaptophysin gene.

Provided herein is a method of reducing caspase-3 protein and/or caspase-3 gene levels in a subject in need thereof, comprising miR-485 A method is provided comprising administering to a subject a compound that inhibits (a miRNA inhibitor). In some aspects, the subject has a disease or condition associated with increased levels of caspase-3 protein and/or caspase-3 gene.

In some aspects, the miR-485 inhibitor that can be used in the above methods induces neurogenesis. In certain aspects, inducing neurogenesis comprises increasing proliferation, differentiation, migration, and/or survival of neural stem and/or progenitor cells. In some aspects, inducing neurogenesis comprises increasing the number of neural stem and/or progenitor cells. In some aspects, inducing neurogenesis comprises increasing axonal, dendrite, and/or synaptic development. In certain aspects, the miR-485 inhibitor induces phagocytosis.

Provided herein is a method of treating a disease or condition associated with abnormal levels of the SIRT1 protein and/or SIRT1 gene in a subject in need thereof, comprising a compound that inhibits miR-485 Also provided are methods comprising administering (a miRNA inhibitor) to a subject, wherein the miRNA inhibitor increases levels of SIRT1 protein and/or SIRT1 gene. Provided herein is a method of treating a disease or condition associated with abnormal levels of CD36 protein and/or CD36 gene in a subject in need thereof, comprising a compound that inhibits miR-485 Also provided are methods comprising administering (a miRNA inhibitor) to a subject, wherein the miRNA inhibitor increases levels of CD36 protein and/or CD36 gene. Provided herein is a method of treating a disease or condition associated with abnormal levels of the PGC-1α protein and/or PGC-1α gene in a subject in need thereof, comprising miR-485 Also provided are methods comprising administering to the subject a compound that inhibits (miRNA inhibitor), wherein the miRNA inhibitor increases levels of PGC-1α protein and/or PGC-1α gene. Provided herein is a method of treating a disease or condition associated with aberrant levels of the LRRK2 protein and/or LRRK2 gene in a subject in need thereof, comprising a compound that inhibits miR-485 Also provided are methods comprising administering (a miRNA inhibitor) to a subject, wherein the miRNA inhibitor increases levels of LRRK2 protein and/or LRRK2 gene. Provided herein is a method of treating a disease or condition associated with abnormal levels of NRG1 protein and/or NRG1 gene in a subject in need thereof, comprising a compound that inhibits miR-485 Also provided are methods comprising administering (a miRNA inhibitor) to a subject, wherein the miRNA inhibitor increases levels of NRG1 protein and/or NRG1 gene. Provided herein is a method of treating a disease or condition associated with aberrant levels of the STMN2 protein and/or the STMN2 gene in a subject in need thereof, comprising a compound that inhibits miR-485 Also provided are methods comprising administering (a miRNA inhibitor) to a subject, wherein the miRNA inhibitor increases STMN2 protein and/or STMN2 gene levels. Provided herein is a method of treating a disease or condition associated with aberrant levels of the VLDLR protein and/or VLDLR gene in a subject in need thereof, comprising a compound that inhibits miR-485 Also provided are methods comprising administering (a miRNA inhibitor) to a subject, wherein the miRNA inhibitor increases VLDLR protein and/or VLDLR gene levels. Provided herein is a method of treating a disease or condition associated with aberrant levels of the NRXN1 protein and/or NRXN1 gene in a subject in need thereof, comprising a compound that inhibits miR-485 Also provided are methods comprising administering (a miRNA inhibitor) to a subject, wherein the miRNA inhibitor increases levels of NRXN1 protein and/or NRXN1 gene. Provided herein is a method of treating a disease or condition associated with aberrant levels of the GRIA4 protein and/or GRIA4 gene in a subject in need thereof, comprising a compound that inhibits miR-485 Also provided are methods comprising administering (a miRNA inhibitor) to a subject, wherein the miRNA inhibitor increases levels of GRIA4 protein and/or GRIA4 gene. Provided herein is a method of treating a disease or condition associated with aberrant levels of the NXPH1 protein and/or NXPH1 gene in a subject in need thereof, comprising a compound that inhibits miR-485 Also provided are methods comprising administering (a miRNA inhibitor) to a subject, wherein the miRNA inhibitor increases levels of NXPH1 protein and/or NXPH1 gene. Provided herein is a method of treating a disease or condition associated with aberrant levels of the PSD-95 protein and/or PSD-95 gene in a subject in need thereof, comprising miR-485 Also provided are methods comprising administering to the subject a compound that inhibits (miRNA inhibitor), wherein the miRNA inhibitor increases levels of PSD-95 protein and/or PSD-95 gene. Provided herein is a method of treating a disease or condition associated with aberrant levels of synaptophysin protein and/or synaptophysin gene in a subject in need thereof, comprising a compound that inhibits miR-485 Also provided are methods comprising administering (a miRNA inhibitor) to a subject, wherein the miRNA inhibitor increases levels of synaptophysin protein and/or synaptophysin gene. Provided herein is a method of treating a disease or condition associated with aberrant levels of caspase-3 protein and/or caspase-3 gene in a subject in need thereof, comprising miR-485 Also provided are methods comprising administering to the subject a compound that inhibits (miRNA inhibitor), wherein the miRNA inhibitor reduces levels of caspase-3 protein and/or caspase-3 gene.

In some aspects, the miRNA inhibitor inhibits miR485-3p. In some aspects, miR485-3p comprises 5'-GUCAUACACGGCUCUCCUCCUCU-3' (SEQ ID NO: 1). In some aspects, the miRNA inhibitor comprises a nucleotide sequence comprising 5'-UGUAUGA-3' (SEQ ID NO:2), and the miRNA inhibitor is about 6 to about 30 nucleotides in length.

In some aspects, the miRNA inhibitor increases SIRT1 gene transcription and/or SIRT1 protein expression, increases CD36 gene transcription and/or CD36 protein expression, PGC1 gene transcription and/or Increase expression of PGC1 protein, increase transcription of LRRK2 gene and/or expression of LRRK2 protein, increase transcription of NRG1 gene and/or expression of NRG1 protein, or increase transcription of STMN2 gene and/or STMN2 protein increase the expression of VLDLR gene transcription and / or VLDLR protein expression, increase NRXN1 gene transcription and / or NRXN1 protein expression, GRIA4 gene transcription and / or GRIA4 protein expression or increase the transcription of the NXPH1 gene and / or the expression of the NXPH1 protein, the transcription of the PSD-95 gene and / or the expression of the PSD-95 protein, the transcription of the synaptophysin gene and / or the synaptophysin protein or decrease caspase-3 gene transcription and/or caspase-3 protein expression, or any combination thereof.

In some aspects, the miRNA inhibitor has at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at the 5' end of the nucleotide sequence. nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides contains nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides. In some aspects, the miRNA inhibitor has at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at the 3' end of the nucleotide sequence. nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides contains nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides.

In some aspects, the miRNA inhibitor is 5′-UGUAUGA-3′ (SEQ ID NO:2), 5′-GUGUAUGA-3′ (SEQ ID NO:3), 5′-CGUGUAUGA-3′ (SEQ ID NO:4), 5′-CCGUGUAUGA-3′ (SEQ ID NO: 5), 5′-GCGUGUAUGA-3′ (SEQ ID NO: 6), 5′-AGCCGUGUAUGA-3′ (SEQ ID NO: 7), 5′-GAGCCGUGAUGA-3′ (SEQ ID NO: 8 ), 5′-AGAGCCGUGUAUGA-3′ (SEQ ID NO: 9), 5′-GAGAGCGUGUAUGA-3′ (SEQ ID NO: 10), 5′-GGAGAGCCGUGUAUGA-3′ (SEQ ID NO: 11), 5′-AGGAGAGCCGGUGUAUGA-3′ (sequence No. 12), 5′-GAGGAGAGCCGUGUAUGA-3′ (SEQ ID NO: 13), 5′-AGAGGAGAGCCGGUAUGA-3′ (SEQ ID NO: 14), and 5′-GAGAGGAGAGCGUGUAUGA-3′ (SEQ ID NO: 15) has an array.

In some aspects, the miRNA inhibitor is 5′-UGUAUGAC-3′ (SEQ ID NO: 16), 5′-GUGUAUGAC-3′ (SEQ ID NO: 17), 5′-CGUGUAUGAC-3′ (SEQ ID NO: 18), 5′-CCGUGUAUUGAC-3′ (SEQ ID NO: 19), 5′-GCGUGUAUUGAC-3′ (SEQ ID NO: 20), 5′-AGCCGUGUAUGAC-3′ (SEQ ID NO: 21), 5′-GAGCCGUGUAUGAC-3′ (SEQ ID NO: 22 ), 5′-AGAGCCGUGUAUGAC-3′ (SEQ ID NO: 23), 5′-GAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 24), 5′-GGAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 25), 5′-AGGAGAGCCGUGUAUGAC-3′ (sequence No. 26), 5′-GAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 27), 5′-AGAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 28), 5′-GAGAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 29), and AGAGAGGAGAGCCGUGUAUGAC (SEQ ID NO: 30) having a sequence selected from the group consisting of

In some aspects, the miRNA inhibitor is 5′-TGTATGA-3′ (SEQ ID NO:62), 5′-GTGTATGA-3′ (SEQ ID NO:63), 5′-CGTGTATGA-3′ (SEQ ID NO:64), 5′-CCGTGTATGA-3′ (SEQ ID NO: 65), 5′-GCCGTGTATGA-3′ (SEQ ID NO: 66), 5′-AGCCGTGTATGA-3′ (SEQ ID NO: 67), 5′-GAGCCGTGTATGA-3′ (SEQ ID NO: 68 ), 5′-AGAGCCGTGTATGA-3′ (SEQ ID NO: 69), 5′-GAGAGCCGTGTATGA-3′ (SEQ ID NO: 70), 5′-GGAGAGCCGTGTATGA-3′ (SEQ ID NO: 71), 5′-AGGAGAGCCGTGTATGA-3′ (sequence No. 72), 5′-GAGGAGAGCCGTGTATGA-3′ (SEQ ID NO: 73), 5′-AGAGGAGAGCCGTGTATGA-3′ (SEQ ID NO: 74), 5′-GAGAGGAGAGCCGTGTATGA-3′ (SEQ ID NO: 75), 5′-TGTATGAC-3′ (SEQ ID NO: 76), 5′-GTGTATGAC-3′ (SEQ ID NO: 77), 5′-CGTGTATGAC-3′ (SEQ ID NO: 78), 5′-CCGTGTATGAC-3′ (SEQ ID NO: 79), 5′-GCCGTGTATGAC- 3′ (SEQ ID NO: 80), 5′-AGCCGTGTATGAC-3′ (SEQ ID NO: 81), 5′-GAGCCGTGTATGAC-3′ (SEQ ID NO: 82), 5′-AGAGCCGTGTATGAC-3′ (SEQ ID NO: 83), 5′- GAGAGCCGTGTATGAC-3′ (SEQ ID NO: 84), 5′-GGAGAGCCGTGTATGAC-3′ (SEQ ID NO: 85), 5′-AGGAGAGCCGTGTATGAC-3′ (SEQ ID NO: 86), 5′-GAGGAGAGCCGTGTATGAC-3′ (SEQ ID NO: 87), 5 '-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:88), 5'-GAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:89), and 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:90).

In some aspects, the sequence of the miRNA inhibitor is 5′-AGAGAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO:30) or 5′-AGAGAGGAGAGCCGTGTATGAC-3′ (SEQ ID NO:90) with at least about 50%, at least about 55%, have at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity. In certain aspects, the miRNA inhibitor has at least 90% similarity with 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:30) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:90). In some aspects, the miRNA inhibitor has a nucleotide sequence 5′-AGAGAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO:30) or 5′-AGAGAGGAGAGCCGTGTATGAC-3′ (SEQ ID NO:90) with one substitution or two substitutions. include. In some aspects, the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:30) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:90). In some aspects, the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:30).

In some aspects, the miRNA inhibitor comprises at least one modified nucleotide. In some aspects, the at least one modified nucleotide is a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), an arabinonucleic acid (ABA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA).

In some aspects, the miRNA inhibitor comprises a backbone modification. In certain aspects, backbone modifications are phosphorodiamidite morpholino oligomers (PMO) and/or phosphorothioate (PS) modifications.

In some aspects, the miRNA inhibitor is delivered in a delivery agent. In certain aspects, the delivery agent is a micelle, exosome, lipid nanoparticle, extracellular vesicle, or synthetic vesicle.

In some aspects, the miRNA inhibitor is delivered by a viral vector. In particular aspects, the viral vector is AAV, adenovirus, retrovirus, or lentivirus. In some aspects, the viral vector is AAV with a serotype of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or any combination thereof.

In some aspects, the miRNA inhibitor is delivered by a delivery agent. In certain aspects, delivery agents comprise lipidoids, liposomes, lipoplexes, lipid nanoparticles, polymeric compounds, peptides, proteins, cells, nanoparticle mimetics, nanotubes, or conjugates.

In some embodiments, the delivery agent has the formula:
[WP]-L1-[CC]-L2-[AM] (Formula I)
or [WP]-L1-[AM]-L2-[CC] (Formula II)
(In the formula,
WP is the water-soluble biopolymer moiety;
CC is a positively charged carrier moiety,
AM is the adjuvant moiety;
L1 and L2 are independently optional linkers, comprising a cationic carrier unit with
Cationic carrier units form micelles when mixed with nucleic acids in an ionic ratio of about 1:1.

In some aspects, miRNA inhibitors interact with cationic carrier units via ionic bonds. In some aspects, the water-soluble polymer is poly(alkylene glycol), poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate) , poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazoline (“POZ”), poly(N-acryloylmorpholine), or any combination thereof . In other aspects, the water-soluble polymer comprises polyethylene glycol (“PEG”), polyglycerol, or poly(propylene glycol) (“PPG”).

In some aspects, the water-soluble polymer has the formula:

を有する。

have

In some aspects, n is 1-1000. In particular aspects, n is at least about 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about 121, at least about 122, at least about 123, at least about 124, at least about 125, at least about 126, at least about 127, at least about 128, at least about 129, at least about 130, at least about 131, at least about 132, at least about 133, at least about 134, at least about 135, at least about 136, at least about 137, at least about 138, at least about 139, at least about 140, or at least about 141; In further aspects, n is from about 80 to about 90, from about 90 to about 100, from about 100 to about 110, from about 110 to about 120, from about 120 to about 130, from about 140 to about 150, or from about 150 to about 160. be.

In some aspects, the water-soluble polymer is linear, branched, or dendritic.

In some aspects, the cationic carrier moiety comprises one or more basic amino acids. In particular aspects, the cationic carrier moieties are at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 , at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37 , at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or Contains at least 50 basic amino acids. In certain aspects, the cationic carrier moiety comprises from about 30 to about 50 basic amino acids.

In some aspects, basic amino acids include arginine, lysine, histidine, or any combination thereof. In some aspects, the cationic carrier moiety comprises about 40 lysine monomers.

In some aspects, an adjuvant moiety can modulate an immune response, an inflammatory response, and/or a tissue microenvironment. In certain aspects, the adjuvant moiety comprises imidazole derivatives, amino acids, vitamins, or any combination thereof.

In some embodiments, the adjuvant moiety has the formula:

(wherein each of G1 and G2 is H, an aromatic ring, or 1-10 alkyl, or G1 and G2 together form an aromatic ring and n is 1-10).

In some aspects, the adjuvant moiety comprises a nitroimidazole. In certain aspects, the adjuvant moiety comprises metronidazole, tinidazole, nimorazole, dimetridazole, pretomanide, ornidazole, megazole, azanidazole, benznidazole, or any combination thereof.

In some aspects, the adjuvant moiety comprises an amino acid.

In some embodiments, the adjuvant moiety has the formula:

であり、 (wherein Ar is

and

each of Z1 and Z2 is H or OH.

In some aspects, the adjuvant portion includes vitamins. In certain aspects, the vitamin comprises a cyclic ring or cyclic heteroatom ring and a carboxyl or hydroxyl group.

In some embodiments, the vitamin has the formula:

where each of Y1 and Y2 is C, N, O, or S and n is 1 or 2.

In some aspects, the vitamins are vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D2, vitamin D3, vitamin E, vitamin M, vitamin H , and any combination thereof. For example, the vitamin can be vitamin B3.

In some aspects, the adjuvant moieties are at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or containing at least about 20 vitamin B3. In certain aspects, the adjuvant portion comprises about 10 vitamin B3.

In some aspects, the delivery agent comprises a water-soluble biopolymer moiety having about 120 to about 130 PEG units, a cationic carrier moiety comprising polylysine having about 30 to about 40 lysines, and about and an adjuvant moiety having 5 to about 10 vitamin B3.

In some aspects, delivery agents are conjugated to miRNA inhibitors to form micelles. For example, binding may be covalent, non-covalent, or ionic.

In some aspects, the cationic carrier unit and the miRNA inhibitor in the micelle are in solution such that the ionic ratio of the positive charge of the cationic carrier unit to the negative charge of the miRNA inhibitor is about 1:1. mixed. In some aspects, the cationic carrier unit can protect the miRNA inhibitor from enzymatic degradation.

In some aspects, diseases or conditions that can be treated according to the present disclosure include Alzheimer's disease. In certain aspects, the disease or condition comprises an autism spectrum disorder, mental retardation, epileptic seizures, stroke, Parkinson's disease, spinal cord injury, or any combination thereof. In certain aspects, the disease or condition is Parkinson's disease.

In some aspects, the delivery agent is a micelle. In some aspects, the micelles comprise (i) about 100 to about 200 PEG units, (ii) about 30 to about 40 lysines each having an amine group, (iii) each having a thiol group. and (iv) about 30 to about 40 lysines each bound to vitamin B3. In some aspects, the micelles comprise (i) about 120 to about 130 PEG units, (ii) about 32 lysines each having an amine group, (iii) about 16 lysines each having a thiol group. and (iv) approximately 32 lysines each bound to vitamin B3.

In some aspects, a targeting moiety is further attached to the PEG unit. In some aspects, the targeting moiety is a LAT1 targeting ligand. In some aspects, the targeting moiety is phenylalanine.

4 shows an exemplary structure of a carrier unit of the present disclosure; Examples presented include anionic payloads, eg, cationic carrier moieties that can electrostatically interact with nucleic acids such as gene-targeted antisense oligonucleotides, eg, miRNAs (antimirs). In some aspects, the AM may be placed between the WP and the CC. The CC and AM components are drawn in a simple linear arrangement. However, as illustrated in FIG. 4, the CC and AM can be arranged in a scaffold.

SIRT1 expression is decreased in subjects with Alzheimer's disease. Shown is a comparison of representative SIRT1 protein expression in the central gyrus from normal patients (ie, subjects without AD) and AD patients (each group, n=6). SIRT1 expression is decreased in subjects with Alzheimer's disease. Figure 2A shows a quantitative comparison of the results shown in Figure 2A. SIRT1 bands were analyzed by densitometry and normalized to β-actin. Relative levels of SIRT1 protein from control (n=6) or AD (n=6) central precentral tissue are shown. Bars indicate mean±SD. SIRT1 expression is decreased in subjects with Alzheimer's disease. SIRT1 mRNA in 6-month-old wild-type (WT) mice (n=4), 6-month-old 5XFAD mice (n=3), 11-month-old wild-type mice (WT), and 11-month-old 5XFAD mice (n=3). shows a comparison of the expression of Comparative analysis was performed on mice of the same age. Bars indicate mean±SD. SIRT1 expression is decreased in subjects with Alzheimer's disease. A comparison of SIRT1 mRNA in 5XFAD mice by age is shown. 5xFAD expression for each age group was normalized to WT. Bars indicate mean±SD.

(A) and (B) show a comparison of miR485-3p and miR485-5p expression in normal patients (ie, subjects without AD) and AD patients, respectively.

A comparison of relative levels of mouse miR485-3p expression in primary cortical neurons transfected with either control oligonucleotides or miR485 inhibitors is shown. The graph on the left shows the expression of miR485-3p after treatment with miR485-3p ASO (also referred to herein as "miRNA inhibitor" or "miR485 inhibitor") for 3 hours. The graph on the right shows expression after treatment with miR485-3p ASO for 6 hours. In each graph, the left bar represents the control group and the right bar represents the miR-485 inhibitor transfected group.

Figure 2 shows that miR-485 inhibitors can increase the expression of SIRT1 and PGC-1α. (A) SIRT1 and PGC-1α protein expression in mouse primary cortical neurons transfected with miR control, miR485-3p (“miR485-3p mimetic”), or miR-485 inhibitor (“miR485-3p ASO”). Western blot results showing . (B) shows a quantitative comparison of the results shown in (A).

miR-485 inhibitors functionally bind to the 3'UTR of SIRT1. Schematic diagram of wild-type (WT) or mutant (MT) in the 3'UTR of SIRT1 showing putative miR-485-3p target sites. Figure 2 shows that miR-485 inhibitors functionally bind to the UTR of SIRT1. Shown is a comparison of relative luciferase activity in HEK293T cells co-transfected with WT or MT reporter constructs of the 3'UTR of SIRT1 and the miR control, miR-485-3p, for 48 hours. At least three independent experiments were performed. Figure 2 shows that miR-485 inhibitors functionally bind to the UTR of SIRT1. A comparison of the relative binding of miR485-3p onto the 3'UTR of SIRT1 with mutant seed region compared to the WT 3'UTR of SIRT1 is shown.

We show that miR-485 inhibitors alter Aβ deposition and APP processing. Shown is the schedule for ICV injection of miR-485 inhibitors in 10 month old 5XFAD mice. We show that miR-485 inhibitors alter Aβ deposition and APP processing. Representative immunohistochemical staining for Aβ(6E10) in cortical and hippocampal DG regions from control (n=5) and 5XFAD mice injected with miR-485 inhibitor (“miR485-3p ASO”) (n=5). target image. We show that miR-485 inhibitors alter Aβ deposition and APP processing. A quantitative comparison of the results shown in FIG. 7B (average number of Aβ plaques per mm ² ) is shown. We show that miR-485 inhibitors alter Aβ deposition and APP processing. Shown are immunoblots of the insoluble Aβ fraction in control (n=3) and 10-month-old 5XFAD mice injected with a miR-485 inhibitor (“miR485-3p ASO”) (n=3). We show that miR-485 inhibitors alter Aβ deposition and APP processing. Figure 7D shows a quantitative comparison of the data shown in Figure 7D. The left bar represents the control group and the right bar represents the miR-485 inhibitor group. We show that miR-485 inhibitors alter Aβ deposition and APP processing. APP, sAPPβ, sAPPα, β-CTFs, BACE1, Adam10, SIRT1 and 10-month-old 5XFAD mice injected with control (n=3) or miR-485 inhibitor (“miR485-3p ASO”) (n=3) Western blots showing expression of PGC-1α protein are shown. We show that miR-485 inhibitors alter Aβ deposition and APP processing. Figure 7F shows a quantitative comparison (ie, relative levels) of the data shown in Figure 7F. In each graph shown in FIG. 7G, the left bar represents the control and the right bar represents the miR-485 inhibitor group.

We show that miR-485 inhibitors enhance Aβ phagocytosis both in vitro and in vivo by increasing CD36 expression. Iba1 in coronal sections of 5XFAD mice injected with control (n=11 images from 5 mice) or miR485-3p ASO (“miR485-3p ASO”) (n=11 images from 5 mice) (microglia) and β-amyloid 1-16 (6E10, for detecting Aβ plaques). We show that miR-485 inhibitors enhance Aβ phagocytosis both in vitro and in vivo by increasing CD36 expression. Quantitative comparison (mean number of Iba1 ⁺ Aβ ⁺ cells per mm ² ) of the data presented in FIG. 8A is shown. We show that miR-485 inhibitors enhance Aβ phagocytosis both in vitro and in vivo by increasing CD36 expression. Hippocampus and cortex of mice treated with control (n=7 images from 3 mice) or miR-485 inhibitor (“miR485-3p ASO”) (n=7 images from 3 mice). Representative images of ThS staining for Aβ plaques are shown. We show that miR-485 inhibitors enhance Aβ phagocytosis both in vitro and in vivo by increasing CD36 expression. Figure 8C shows a quantitative comparison of the data shown in Figure 8C. We show that miR-485 inhibitors enhance Aβ phagocytosis both in vitro and in vivo by increasing CD36 expression. Immunohistochemical analysis showing uptake of Aβ plaques (Aβ1-42) by primary glial cells (Iba1+) in mouse primary mixed glial cells transfected and/or treated with one of the following: (i) control oligos; (ii) treated with fAβ(1-42) (1 μM), or (iii) transfected with a miR-485 inhibitor (“miR485-3p ASO”) and fAβ(1-42) (1 μM). We show that miR-485 inhibitors enhance Aβ phagocytosis both in vitro and in vivo by increasing CD36 expression. Control (n=6) or miR-485 inhibitor (“miR485-3p ASO”) (n=6) injections with anti-Iba1, anti-CD68 (phagosome), and anti-β-amyloid 1-16 (6E10) Immunohistochemical analyzes of histological brain sections from 5XFAD mice are shown. We show that miR-485 inhibitors enhance Aβ phagocytosis both in vitro and in vivo by increasing CD36 expression. Quantitative comparison (mean number of Iba1 ⁺ Aβ ⁺ CD68 ⁺ cells per mm ² ) of the results shown in FIG. 8F is shown. We show that miR-485 inhibitors enhance Aβ phagocytosis both in vitro and in vivo by increasing CD36 expression. Comparison of Aβ levels in supernatants of BV2 microglial cells transfected with either control oligonucleotides or miR-485 inhibitors (“miR485-3p ASO”) and treated with fAβ(1-42) (1 μM). indicates Supernatants were collected 4 hours after treatment and analyzed by ELISA.

Figure 2 shows that miR-485 inhibitors can increase CD36 expression. A comparison of the relative levels of CD36 protein expression in control (n=3) and 10-month-old 5XFAD mice injected with a miR-485 inhibitor (“miR485-3p ASO”) (n=3) is shown. Figure 2 shows that miR-485 inhibitors can increase CD36 expression. Figure 9A shows a quantitative comparison of the results shown in Figure 9A. Figure 2 shows that miR-485 inhibitors can increase CD36 expression. Immunohistochemical analysis of histological brain sections from 5XFAD mice treated with control or miR-485 inhibitor (“miR485-3p ASO”) using anti-Iba1 and anti-β amyloid 1-16 (6E10). . Figure 2 shows that miR-485 inhibitors can increase CD36 expression. Using Alexa488-conjugated anti-CD36 antibody in primary mixed glial cells transfected with control (n=3), miR-485-3p mimic, or miR-485 inhibitor (“miR485-3p ASO”) (n=3). Cell surface expression of CD36 measured by flow cytometry is shown. Figure 2 shows that miR-485 inhibitors can increase CD36 expression. FIG. 9D shows a quantitative comparison (relative mean fluorescence intensity) of the results shown in FIG. 9D.

Figure 2 shows that miR-485 inhibitors can functionally bind to the 3'UTR of CD36. Relative luciferase activity was measured in HEK293T cells co-transfected with WT or MT reporter constructs of CD36 3'UTR and miR control or miR-485 inhibitor for 48 hours.

We show that miR-485 inhibitors can promote increased Aβ phagocytosis through modulation of CD36. Aβ levels in supernatants of BV2 microglial cells transfected with either control oligonucleotides or a miR-485 inhibitor (“miR485-3p ASO”) and treated with fAβ(1-42) (1 μM). Blocking anti-CD36 antibodies were also added where indicated. Supernatants were collected 4 hours after treatment and analyzed by ELISA.

Figure 4 shows that miR-485 inhibitors can reduce glial neuroinflammation. SIRT1, NF-κB(p65) in primary mixed glial cells transfected with control or miR-485 inhibitor (“miR485-3p ASO”) treated with fAβ(1-42) (1 μM) for 3-6 h , Western blots showing expression of TNF-α and IL-1β proteins. "(1)" corresponds to cells transfected with control oligonucleotide alone. "(2)" corresponds to cells treated with fAβ(1-42) alone. "(3)" corresponds to cells transfected with miR-485 inhibitors and treated with fAβ(1-42). Figure 4 shows that miR-485 inhibitors can reduce glial neuroinflammation. Figure 12A shows a quantitative comparison of the results shown in Figure 12A. In each of the graphs shown in FIG. 12B, the left bar represents control, the middle bar represents cells treated with fAβ(1-42) alone, the right bar transfected with miR-485 inhibitor, Cells treated with fAβ(1-42) are shown. Figure 4 shows that miR-485 inhibitors can reduce glial neuroinflammation. Iba1, NF-κB(p65), TNF-α and IL-1b in control (n=3) and 10 month old 5XFAD mice injected with a miR-485 inhibitor (“miR485-3p ASO”) (n=3) Immunoblot detection of proteins is shown. Results from two independent experiments are shown (ie, set 1 and set 2). Figure 4 shows that miR-485 inhibitors can reduce glial neuroinflammation. Figure 12C shows a quantitative comparison of the results shown in Figure 12C. In each graph shown in Figure 12D, the left bar represents the control and the right bar represents the miR-485 inhibitor group. Figure 4 shows that miR-485 inhibitors can reduce glial neuroinflammation. Iba1 and TNF in 5XFAD mice injected with control (n=11, images from 5 mice) or miR-485 inhibitor (“miR485-3p ASO”) (n=11, images from 5 mice) - Immunohistochemical analysis of the expression of α. Figure 4 shows that miR-485 inhibitors can reduce glial neuroinflammation. A quantitative comparison (mean number of Iba1 and TNF-α stained cells per mm ² ) of the results shown in FIG. 12E is shown. Figure 4 shows that miR-485 inhibitors can reduce glial neuroinflammation. Iba1 and IL in 5XFAD mice injected with control (n=11, images from 5 mice) or miR-485 inhibitor (“miR485-3p ASO”) (n=11, images from 5 mice) Immunohistochemical analysis of -1β expression is shown. Figure 4 shows that miR-485 inhibitors can reduce glial neuroinflammation. A quantitative comparison (mean number of Iba1 and IL-1β stained cells per mm ² ) of the results shown in FIG. 12G is shown. Figure 4 shows that miR-485 inhibitors can reduce glial neuroinflammation. Amount of TNF-α observed in supernatants of primary mixed glial cells treated with mPFF (mouse α-synuclein aggregate, 1 μg/mL) and different concentrations of miR-485 inhibitors (50 and 100 nM) (FIG. 12I) shows a comparison of Figure 4 shows that miR-485 inhibitors can reduce glial neuroinflammation. A comparison of the amount of IL-1β observed in supernatants of primary mixed glial cells treated with mPFF (mouse α-synuclein aggregated, 1 μg/mL) and different concentrations of miR-485 inhibitors (50 and 100 nM) is shown. .

We show that miR-485 inhibitors ameliorate neuronal cell loss, promote neurogenesis, and increase postsynaptic hyperplasia. NeuN and cleaved caspases in hippocampus (left) and cortex (right) of 10-month-old 5XFAD mice injected with control (n=3) or miR-485 inhibitor (“miR485-3p ASO”) (n=5). An immunoblot showing the expression of 3 proteins is shown. We show that miR-485 inhibitors ameliorate neuronal cell loss, promote neurogenesis, and increase postsynaptic hyperplasia. Figure 13A shows a quantitative comparison of the results shown in Figure 13A. We show that miR-485 inhibitors ameliorate neuronal cell loss, promote neurogenesis, and increase postsynaptic hyperplasia. Expression of NeuN and cleaved caspase-3 in coronal brain sections from 10-month-old 5XFAD mice injected with control (n=4) or miR-485 inhibitor (“miR485-3p ASO”) (n=5) was examined. Immunohistochemical analysis is shown. We show that miR-485 inhibitors ameliorate neuronal cell loss, promote neurogenesis, and increase postsynaptic hyperplasia. A quantitative comparison (average number of NeuN and cleaved caspase-3 stained cells per mm ² ) of the results shown in FIG. 13C is shown. We show that miR-485 inhibitors ameliorate neuronal cell loss, promote neurogenesis, and increase postsynaptic hyperplasia. Immunoblot analysis of PSD-95 protein expression in control (n=3) and 10 month old 5XFAD mice injected with miR-485 inhibitor (“miR485-3p ASO”) (n=5) is shown. Results from two independent experiments are shown (ie, set 1 and set 2). We show that miR-485 inhibitors ameliorate neuronal cell loss, promote neurogenesis, and increase postsynaptic hyperplasia. Figure 13E shows a quantitative comparison of the results shown in Figure 13E. We show that miR-485 inhibitors ameliorate neuronal cell loss, promote neurogenesis, and increase postsynaptic hyperplasia. A comparison of doublecortin (DCX) positive cells in brain tissue from control mice or 5XFAD mice treated with miR-485 inhibitors is shown.

(A) and (B) show that miR-485 inhibitors ameliorate cognitive decline in 5XFAD mice. Y-maze test and passive avoidance test results are shown for mice (10 month old 5XFAD mice) treated with either control oligonucleotides or miR-485 inhibitors (“miR485-3p ASO”), respectively. Mean alternation rate (%) and total number of entries into each arm of the Y-maze of 5XFAD mice injected with control or miR485-3p. Mean locomotion latency and time in the dark (sec) of control or miR-485-3p-injected 5XFAD mice in the passive avoidance test. (A) and (B) show that miR-485 inhibitors ameliorate cognitive decline in 5XFAD mice. Y-maze test and passive avoidance test results are shown for mice (10 month old 5XFAD mice) treated with either control oligonucleotides or miR-485 inhibitors (“miR485-3p ASO”), respectively. Mean alternation rate (%) and total number of entries into each arm of the Y-maze of 5XFAD mice injected with control or miR485-3p. Mean locomotion latency and time in the dark (sec) of control or miR-485-3p-injected 5XFAD mice in the passive avoidance test.

Schematic representation of different possible non-limiting means by which miR-485 inhibitors can treat Alzheimer's disease, demonstrated using 5XFAD mice. In 5XFAD, miR-485 inhibitors can increase neuronal SIRT1 expression. SIRT1 can then reduce amyloid-beta production through regulation of amyloidogenic enzymes. Inhibitors of miR-485 can also promote CD36 expression in glial cells and phagocytosis of Aβ plaques. At the same time, miR-485 inhibitors can induce the expression of SIRT1 and reduce neuroinflammation and nerve damage.

Figure 2 shows increased expression of SIRT1 and PGC-1α after a single intracerebroventricular administration of miR-485 inhibitors in the brain cortex of mice. (A) shows the expression levels of SIRT1 (left graph) and PGC-1α (right graph) 6, 24, 48, and 72 hours after administration of miR-485 inhibitors (100 μg/mouse). (B) and (C) respectively show a positive correlation between SIRT1 and PGC-1α expression and time over about 50 hours. In each of (A), (B), and (C), SIRT1 and PGC-1α expression is shown normalized to controls (ie, expression levels in mice not treated with miR-485 inhibitors). It is Percent values shown in (A) represent the mean percentage increase in SIRT1 and PGC-1α expression relative to controls 48 hours after miR-485 inhibitor administration. In (A), p-values shown represent t-test p-values. In (B) and (C) the p-values shown represent the p-values of the Pearson correlation. "C.C" represents the correlation coefficient of the Pearson correlation.

Figure 2 shows increased expression of SIRT1 and PGC-1α in the hippocampus of mouse brain after a single intravenous administration of miR-485 inhibitors. (A) shows the expression levels of SIRT1 (left graph) and PGC-1α (right graph) 6, 24, 48, and 72 hours after administration of miR-485 inhibitors (100 μg/mouse). (B) and (C) respectively show a positive correlation between the expression of SIRT1 and PGC-1α and time over approximately 24 hours. In each of (A), (B), and (C), expression levels of SIRT1 and PGC-1α were normalized to controls (ie, expression levels in mice not treated with miR-485 inhibitors). It is shown. Percentage values shown in (A) represent the mean percentage increase in SIRT1 and PGC-1α expression relative to controls 24 hours after miR-485 inhibitor administration. In (A), p-values shown represent t-test p-values. In (B) and (C) the p-values shown represent the p-values of the Pearson correlation. "C.C" represents the correlation coefficient of the Pearson correlation.

CD36 expression is increased after a single intravenous administration of miR-485 inhibitors (100 μg/mouse) in mouse brain. (A) shows CD36 expression levels 24, 48, 72, and 120 hours after miR-485 inhibitor administration (100 μg/mouse). (B) shows a positive correlation between CD36 expression and time over approximately 80 hours. In each of (A) and (B), CD36 expression is shown normalized to controls (ie, expression levels in mice not treated with miR-485 inhibitors). Percentage values shown in (A) represent the mean percentage increase in CD36 expression relative to controls 48 hours after miR-485 inhibitor administration. In (A), p-values shown represent t-test p-values. In (B), p-values indicated represent p-values for Pearson correlation. "C.C" represents the correlation coefficient of the Pearson correlation.

(A) and (B) show that administration of miR-485 inhibitors has no appreciable effect on body weight in male and female rats, respectively. As shown in the figure, male and female rats were given the following doses of miR-485 inhibitors: (i) 0 mg/kg (G1), (ii) 3.75 mg/kg (G2), (iii) ) 7.5 mg/kg (G3), and (iv) 15 mg/kg (G4). Body weights were measured 0, 3, 7, and 14 days after miR-485 inhibitor administration.

(A) and (B) show that administration of miR-485 inhibitors does not affect mortality in male and female rats, respectively. As shown in the figure, male and female rats were given the following doses of miR-485 inhibitors: (i) 0 mg/kg (G1), (ii) 3.75 mg/kg (G2), (iii) ) 7.5 mg/kg (G3), and (iv) 15 mg/kg (G4). Animal mortality was measured daily from days 0-14 after administration of miR-485 inhibitors.

Figure 2 shows that administration of miR-485 inhibitors has no persistent clinical side effects when administered to male rats. As indicated, male rats were given the following doses of miR-485 inhibitors: (i) 0 mg/kg (G1), (ii) 3.75 mg/kg (G2), (iii) 7. 5 mg/kg (G3), and (iv) one of 15 mg/kg (G4). Side effects measured included: (i) NOA (no noticeable abnormalities), (ii) congestion (tail), (iii) edema (face), (iv) edema (forelimb), and (v) edema (hindlimb). Side effects are measured at 0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 1 day, 3 days, 5 days, 8 days, 11 days, and 14 days after administration of miR-485 inhibitors. bottom. Figure 2 shows that administration of miR-485 inhibitors has no persistent clinical side effects when administered to female rats. As indicated, female rats were given the following doses of miR-485 inhibitors: (i) 0 mg/kg (G1), (ii) 3.75 mg/kg (G2), (iii) 7. 5 mg/kg (G3), and (iv) one of 15 mg/kg (G4). Side effects measured included: (i) NOA (no noticeable abnormalities), (ii) congestion (tail), (iii) edema (face), (iv) edema (forelimb), and (v) edema (hindlimb). Side effects are measured at 0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 1 day, 3 days, 5 days, 8 days, 11 days, and 14 days after administration of miR-485 inhibitors. bottom.

(A) and (B) show that miR-485 inhibitor administration has no significant pathological abnormalities in male and female rats, respectively. As shown in the figure, male and female rats were given the following doses of miR-485 inhibitors: (i) 0 mg/kg (G1), (ii) 3.75 mg/kg (G2), (iii) ) 7.5 mg/kg (G3), and (iv) 15 mg/kg (G4).

FIG. 4 shows the therapeutic effect of miR-485 inhibitor administration in a mouse model of Parkinson's disease (ie, 6-OHDA mice). Schematic representation of the experimental design is shown. FIG. 4 shows the therapeutic effect of miR-485 inhibitor administration in a mouse model of Parkinson's disease (ie, 6-OHDA mice). A comparison of rotarod latencies (time required for animals to fall off the rotarod treadmill, as described in the Examples) in 6-OHDA mice treated with PBS or miR-485 inhibitors is shown. FIG. 4 shows the therapeutic effect of miR-485 inhibitor administration in a mouse model of Parkinson's disease (ie, 6-OHDA mice). A comparison of latencies for animals to fall from wire cages is shown in 6-OHDA mice treated with PBS or miR-485 inhibitors. FIG. 4 shows the therapeutic effect of miR-485 inhibitor administration in a mouse model of Parkinson's disease (ie, 6-OHDA mice). A comparison of the time required to descend the pole is shown in 6-OHDA mice treated with PBS or miR-485 inhibitors. FIG. 4 shows the therapeutic effect of miR-485 inhibitor administration in a mouse model of Parkinson's disease (ie, 6-OHDA mice). Shown is a comparison of the number of paw slides (left graph) and the time required to traverse the entire length of the beam in 6-OHDA mice treated with PBS or miR-485 inhibitors. FIG. 4 shows the therapeutic effect of miR-485 inhibitor administration in a mouse model of Parkinson's disease (ie, 6-OHDA mice). The following groups: (i) wild-type mice treated with PBS (control + PBS, first and third columns from left); (ii) 6-OHDA mice treated with PBS (experimental + PBS, second from left); and 4th columns), (iii) miR-485 inhibitor-treated wild-type mice (control + miR-485, 5th and 7th columns from left), and (iv) miR-485 inhibitor-treated Western blot showing expression of tyrosine hydroxylase (TH) in the substantia nigra (SN) and striatum (STR) of 6-OHDA mice (experimental + miR-485, 6th and 8th columns from left), respectively. Show analysis. FIG. 4 shows the therapeutic effect of miR-485 inhibitor administration in a mouse model of Parkinson's disease (ie, 6-OHDA mice). FIG. 23F shows a quantitative comparison (relative TH expression level) of the results shown in FIG. 23F. FIG. 4 shows the therapeutic effect of miR-485 inhibitor administration in a mouse model of Parkinson's disease (ie, 6-OHDA mice). The following groups: (i) wild-type mice treated with PBS (control + PBS, first and third columns from left); (ii) 6-OHDA mice treated with PBS (experimental + PBS, second from left); and 4th columns), (iii) miR-485 inhibitor-treated wild-type mice (control + miR-485, 5th and 7th columns from left), and (iv) miR-485 inhibitor-treated Western blot showing expression of tyrosine hydroxylase (TH) in the substantia nigra (SN) and striatum (STR) of 6-OHDA mice (experimental + miR-485, 6th and 8th columns from left), respectively. Show analysis. FIG. 4 shows the therapeutic effect of miR-485 inhibitor administration in a mouse model of Parkinson's disease (ie, 6-OHDA mice). Figure 23H shows a quantitative comparison (relative TH expression level) of the results shown in Figure 23H. FIG. 4 shows the therapeutic effect of miR-485 inhibitor administration in a mouse model of Parkinson's disease (ie, 6-OHDA mice). Western blot analysis showing the expression of the following proteins measured in the substantia nigra of mice in different treatment groups: TNF-α, IL-1β, Iba1, GFAP and β-actin (control). The different treatment groups were as follows. (i) wild-type mice treated with PBS (control + PBS, 1st and 3rd columns from left), (ii) 6-OHDA mice treated with PBS (experimental + PBS, 2nd and 4th columns from left) , (iii) wild-type mice treated with miR-485 inhibitors (control + miR-485, fifth and seventh columns from left), and (iv) 6-OHDA mice treated with miR-485 inhibitors (experimental +miR-485, 6th and 8th columns from left). FIG. 4 shows the therapeutic effect of miR-485 inhibitor administration in a mouse model of Parkinson's disease (ie, 6-OHDA mice). FIG. 23J shows a quantitative comparison of IL-1β expression shown in FIG. 23J.

(A) and (B) show the effect of miR-485 inhibitors on autophagy in primary cortical neurons and primary mixed glial cells, respectively. (A) Western blot results comparing expression of p62 and LC3B in primary cortical neurons treated with mPFF mouse α-synuclein aggregate (1 μg/mL) and increasing concentrations of miR-485 inhibitors. In (A), the gel on the left shows the results after 24 hours of treatment and the gel on the right shows the results after 48 hours of treatment. (B) Western blot results comparing p62 and LC3B expression in primary mixed glial cells treated with mPFF mouse α-synuclein aggregate (1 μg/mL) and increasing concentrations of miR-485 inhibitors. In each of (A) and (B), the first column (from left) represents untreated cells (i.e., without mPFF or miR-485 inhibitor) and the second column (from left) represents mPFF. represents cells treated with only

(A) and (B) show viral vector injection site and lentivirus-induced overexpression of miR-485-3p in mouse hippocampus, respectively. (A) shows targeted bilateral viral vector injection sites (ie, dentate gyrus (DG) and CA1 in the posterior hippocampus). (B) shows expression of green fluorescent protein (GFP) in DG and CA1 of the retro- and anterior hippocampus.

Schematic of rodent behavioral test for cognitive function and memory. OFT (open field test), Y maze test, NORT (novel object recognition test), and PAT (passive avoidance test) were performed on the indicated days.

(A) and (B) Open field test (OFT) in mice injected with lenti-control vector (n=8) (black bars) or lenti-miR485-3p vector (n=7) (grey bars) ) shows the results. (A) shows total distance traveled (cm) in 30 minutes for mice injected with control or lenti-miR485-3p vector. (B) shows percent central area activity for mice injected with control or lenti-miR485-3p vector. Error bars indicate mean±standard error of the mean (SEM). Statistical significance was determined by performing an unpaired t-test followed by Bonferroni's post hoc statistical test.

(A) and (B) Y-maze test in mice injected with lenti-control vector (n=8) (black bars) or lenti-miR485-3p vector (n=7) (grey bars). shows the results of (A) shows the total number of entries into each arm of the Y-maze and (B) shows the average alternation rate (%) for mice injected with control or lenti-miR485-3p vector. P value = 0.795 Error bars represent mean ± SEM. Statistical significance was determined by an unpaired t-test followed by Bonferroni's post hoc statistical test.

Shown are the results of the novel object recognition test (NORT) in mice injected with lenti-control vector or lenti-miR485-3p vector. A scheme for novel object recognition testing is shown. A detailed description of the experimental scheme is provided in Example 19 (headed "Novel Object Recognition Test"). Shown are the results of the novel object recognition test (NORT) in mice injected with lenti-control vector or lenti-miR485-3p vector. Shows the preference of animals in different treatment groups for novel or familiar objects after short-term memory testing (object x miR485-3p interaction p-value = 0.1288; object p = 0.5287, F(1. 22) = 0.4098; virus p = 0.0143, F(1.22) = 7.075; lenti-control n = 11, lenti-mir485-3p n = 9). Statistical significance was determined by two-way ANOVA and unpaired t-test followed by Bonferroni's post hoc statistical test. N. S. = not significant. Shown are the results of the novel object recognition test (NORT) in mice injected with lenti-control vector or lenti-miR485-3p vector. Shows the preference of animals in different treatment groups for novel or familiar objects at 3 days after placement in the chamber (or the day after object recognition training) (long-term memory test 2) (object x miR485-3p interaction p-value p<0.0001, F(1.26)=33.75, object p=0.0459, F(1.26)=43.18; lenti-control n=8, lenti-mir485 -3p n=7). Statistical significance was determined by two-way ANOVA and unpaired t-test followed by Bonferroni's post hoc statistical test. N. S. = not significant. Shown are the results of the novel object recognition test (NORT) in mice injected with lenti-control vector or lenti-miR485-3p vector. Shows the preference of animals in different treatment groups for novel or familiar objects at 24 days after placement in the chamber (or 22 days after object recognition training) (long-term memory test 3) (object x miR485 −3p interaction p-value p=0.0169, F(1.26)=6.523, object p<0.0001, F(1.26)=37.29; lenti-control n=8, lenti -mir485-3p n=7). Statistical significance was determined by two-way ANOVA and unpaired t-test followed by Bonferroni's post hoc statistical test. N. S. = not significant. Shown are the results of the novel object recognition test (NORT) in mice injected with lenti-control vector or lenti-miR485-3p vector. Figure 29B shows the discrimination index (ability to distinguish between novel and familiar objects) based on the results shown in Figure 29B. Error bars represent mean ± SEM. Statistical significance was determined by two-way ANOVA and unpaired t-test followed by Bonferroni's post hoc statistical test. N. S. = not significant. Shown are the results of the novel object recognition test (NORT) in mice injected with lenti-control vector or lenti-miR485-3p vector. Figure 29C shows the discrimination index (ability to distinguish between novel and familiar objects) based on the results shown in Figure 29C. Error bars represent mean ± SEM. Statistical significance was determined by two-way ANOVA and unpaired t-test followed by Bonferroni's post hoc statistical test. N. S. = not significant. Shown are the results of the novel object recognition test (NORT) in mice injected with lenti-control vector or lenti-miR485-3p vector. Figure 29D shows the discrimination index (ability to distinguish between novel and familiar objects) based on the results shown in Figure 29D. Error bars represent mean ± SEM. Statistical significance was determined by two-way ANOVA and unpaired t-test followed by Bonferroni's post hoc statistical test. N. S. = not significant.

Passive avoidance test (PAT) results, i.e., entry latency (seconds), in mice injected with lenti-control vector (n=8) (black) or lenti-miR485-3p vector (n=7) (grey) are shown. show. P value = 0.18 Error bars represent mean ± SEM. Statistical significance was determined by performing an unpaired t-test followed by Bonferroni's post hoc statistical test.

(A) and (B) show the experimental design and results of amyloid-beta (Aβ) production and spread of Aβ from neuron to neuron. (A) shows the experimental design described in Example 19. (B) Viral expression (3rd and 6th images, respectively) and amyloid beta (2nd and 5th images, respectively) in cells transduced with lenti-control vector or lenti-miR485-3p. Results of immunohistochemical analysis are shown.

Fig. 2 shows results of testing truncated tau (C3) production and spread of truncated tau from neuron to neuron. Immunohistochemistry in cells transduced with lenti-control vector or lenti-miR485-3p tested for viral expression (3rd and 6th images, respectively) and truncated tau production (2nd and 5th images, respectively) Shows the results of the analysis.

(A) and (B) show the results of testing the expression of PSD-95 and synaptophysin proteins, respectively. (A) Transduced with lenti-control vector or lenti-miR485-3p, tested for viral expression (3rd and 6th images, respectively) and PSD-95 protein expression (2nd and 5th images, respectively). The results of immunohistochemical analysis on transfected cells are shown. (B) Transduced with lenti-control vector or lenti-miR485-3p, tested for viral expression (3rd and 6th images, respectively) and synaptophysin protein expression (2nd and 5th images, respectively). Results of immunohistochemical analysis on cells are shown.

Fig. 2 shows the results of testing the expression of cleaved caspase-3 protein. Viral expression (3rd and 6th images, respectively) and cleaved caspase-3 expression (2nd and 5th images, respectively) were tested in cells transduced with lenti-control vector or lenti-miR485-3p. Results of immunohistochemical analysis are shown.

Experimental design (A) and results of testing the expression of a microglial cell-specific marker (ionized calcium-binding adapter protein-1 (Iba-1)) (B) and cleaved caspase-3 protein (C) in mouse primary microglial cells. indicate. (A) shows the experimental design described in Example 19. (B) Transduced with lenti-control vector or lenti-miR485-3p, tested for viral expression (3rd and 6th images, respectively) and Iba-1 expression (2nd and 5th images, respectively). 1 shows the results of immunohistochemical analysis in cells obtained from cytotoxicity. (C) lenti-control vector or lenti-control vector tested for viral expression (3rd and 6th images, respectively) and cleaved caspase-3 expression (2nd and 5th images, respectively) in mouse primary glial cells. - shows the results of immunohistochemical analysis in cells transduced with miR485-3p.

(A) and (B) show the results of testing the expression of glial fibrillary acidic protein (GFAP) and cleaved caspase-3 protein as astrocyte-specific markers in mouse primary astrocytes, respectively. (A) lenti-control vector or lenti-miR485- viral expression (3rd and 6th images respectively) and GFAP expression (2nd and 5th images respectively) in mouse primary astrocytes Results of immunohistochemical analysis in 3p-transduced cells are shown. (B) Lenti-control vector tested for viral expression (3rd and 6th images, respectively) and cleaved caspase-3 protein expression (2nd and 5th images, respectively) in mouse primary astrocytes. or the results of immunohistochemical analysis in cells transduced with lenti-miR485-3p.

The experimental design (A) and the results of testing the expression of a microglial cell-specific marker (ionized calcium-binding adapter protein-1 (Iba-1)) (B) and cleaved caspase-3 protein (C) in human microglial cells. show. (A) shows the experimental design described in Example 19. (B) lenti-control vector or lenti-miR485- viral expression (third and sixth images, respectively) and Iba-1 expression (second and fifth images, respectively) in human microglial cells Results of immunohistochemical analysis in 3p-transduced cells are shown. (C) lenti-control vector or lenti- Results of immunohistochemical analysis in cells transduced with miR485-3p are shown.

(A) and (B) show the results of testing the expression of glial fibrillary acidic protein (GFAP) and cleaved caspase-3 protein as astrocyte-specific markers in human astrocytes, respectively. (A) lenti-control vector or lenti-miR485-3p tested for viral expression (3rd and 6th images, respectively) and GFAP expression (2nd and 5th images, respectively) in human astrocytes. shows the results of immunohistochemical analysis in cells transduced with . (B) The lenti-control vector or Results of immunohistochemical analysis in cells transduced with lenti-miR485-3p are shown.

Shows the therapeutic effect of two different doses (2 mg/kg or 5 mg/kg) of miR-485 inhibitors in a mouse model of Parkinson's disease (ie, 6-OHDA). Healthy animals treated with PBS and 6-OHDA mice were used as controls. (A) shows a comparison of rotarod latencies (time taken for animals to fall off the rotarod treadmill, as described in the Examples). (B) shows a comparison of the times taken to descend the pole. (C) shows a comparison of latencies for animals to fall from wire mesh cages. (D) shows a comparison of the number of foot slips that occurred while traversing the entire length of the beam. In each figure, (1) corresponds to the healthy control group, (2) corresponds to 6-OHDA mice treated with PBS, and (3) miR-485 inhibition at 2.5 mg/kg. (4) corresponds to 6-OHDA mice treated with 5 mg/kg miR-485 inhibitor.

Effect of miR-485 inhibitors on autophagy in BV2 microglial cells. Expression of FOXO3a, LC3-I, and LC3-II proteins in BV2 cells treated with fibrillar amyloid beta (oAβ) and transfected with different doses of miR-485 inhibitors (0 nM, 50 nM, 100 nM, or 300 nM). Comparative Western blot results are shown. Cells that were neither treated with oAβ nor transfected with miR-485 inhibitors were used as controls. Effect of miR-485 inhibitors on autophagy in BV2 microglial cells. Quantitative comparison of FOXO3 protein expression in BV2 cells of treatment groups is shown. In each figure, (1) corresponds to control cells (i.e., neither treated with oAβ nor transfected with miR-485 inhibitor) and (2) BV2 cells treated with oAβ alone. Corresponding cells, (3) correspond to BV2 cells treated with oAβ and 50 nM miR-485 inhibitor, (4) correspond to BV2 cells treated with oAβ and 100 nM miR-485 inhibitor. , (5) correspond to BV2 cells treated with oAβ and 300 nM miR-485 inhibitor. Effect of miR-485 inhibitors on autophagy in BV2 microglial cells. Quantitative comparison of p62 protein expression in BV2 cells of treatment groups is shown. In each figure, (1) corresponds to control cells (i.e., neither treated with oAβ nor transfected with miR-485 inhibitor) and (2) BV2 cells treated with oAβ alone. Corresponding cells, (3) correspond to BV2 cells treated with oAβ and 50 nM miR-485 inhibitor, (4) correspond to BV2 cells treated with oAβ and 100 nM miR-485 inhibitor. , (5) correspond to BV2 cells treated with oAβ and 300 nM miR-485 inhibitor. Effect of miR-485 inhibitors on autophagy in BV2 microglial cells. Quantitative comparison of LC3-II protein expression in BV2 cells of treatment groups is shown. In each figure, (1) corresponds to control cells (i.e., neither treated with oAβ nor transfected with miR-485 inhibitor) and (2) BV2 cells treated with oAβ alone. Corresponding cells, (3) correspond to BV2 cells treated with oAβ and 50 nM miR-485 inhibitor, (4) correspond to BV2 cells treated with oAβ and 100 nM miR-485 inhibitor. , (5) correspond to BV2 cells treated with oAβ and 300 nM miR-485 inhibitor.

The present disclosure relates to the use of miR-485 inhibitors comprising nucleotide sequences encoding non-protein-encoding nucleotide molecules comprising at least one miR-485 binding site. In some aspects, the miRNA binding site(s) can bind endogenous miR-485 that inhibits and/or reduces the expression level of the endogenous SIRT1 protein and/or SIRT1 gene. . In some aspects, binding of endogenous miR-485 to the miRNA binding site(s) can inhibit and/or reduce the expression level of endogenous CD36 protein and/or CD36 gene. . Similarly, in some aspects, binding of endogenous miR-485 to the miRNA binding site(s) can inhibit and/or reduce the expression level of endogenous PGC-1α. In some aspects, binding of endogenous miR-485 to miRNA binding site(s) can inhibit and/or reduce the expression level of endogenous LRRK2 protein and/or LRRK2 gene. . In some aspects, binding of endogenous miR-485 to miRNA binding site(s) can inhibit and/or reduce the expression level of endogenous NRG1 protein and/or NRG1 gene. . In some aspects, binding of endogenous miR-485 to miRNA binding site(s) can inhibit and/or reduce the expression level of endogenous STMN2 protein and/or STMN2 gene. . In some aspects, binding of endogenous miR-485 to the miRNA binding site(s) can inhibit and/or reduce expression levels of endogenous VLDLR proteins and/or VLDLR genes. . In some aspects, endogenous miR-485 binding to the miRNA binding site(s) can inhibit and/or reduce the expression level of the endogenous NRXN1 protein and/or NRXN1 gene. . In some aspects, binding of endogenous miR-485 to the miRNA binding site(s) can inhibit and/or reduce expression levels of endogenous GRIA4 protein and/or GRIA4 gene. . In some aspects, binding of endogenous miR-485 to miRNA binding site(s) can inhibit and/or reduce the expression level of endogenous NXPH1 protein and/or NXPH1 gene. . In some aspects, binding of endogenous miR-485 to miRNA binding site(s) inhibits and/or reduces expression levels of endogenous PSD-95 protein and/or PSD-95 gene. can be made In some aspects, binding of endogenous miR-485 to the miRNA binding site(s) can inhibit and/or reduce expression levels of endogenous synaptophysin protein and/or synaptophysin gene. . In some aspects, binding of endogenous miR-485 to the miRNA binding site(s) promotes and/or increases expression levels of endogenous caspase-3 protein and/or caspase-3 gene. can be done.

Accordingly, in some aspects, the present disclosure provides a method of increasing SIRT1 protein and/or SIRT1 gene levels in a subject in need thereof, comprising miR-485 inhibition It relates to a method comprising administering an agent to a subject. In a further aspect, increasing SIRT1 protein and/or SIRT1 gene levels in a subject is used to treat diseases or conditions (e.g., neurodegenerative diseases and disorders) associated with decreased levels of SIRT1 protein and/or SIRT1 gene. can be useful. In some aspects, the disclosure provides a method of increasing CD36 protein and/or CD36 gene levels in a subject in need thereof, comprising a miR-485 inhibitor. It relates to a method comprising administering to a subject. In a further aspect, increasing levels of CD36 protein and/or CD36 gene in a subject is used to treat diseases or conditions associated with decreased levels of CD36 protein and/or CD36 gene (e.g., neurodegenerative diseases and disorders). can be useful. In some aspects, the disclosure is a method of increasing levels of PGC-1α protein and/or PGC-1α gene in a subject in need thereof. and methods comprising administering a miR-485 inhibitor to a subject. In a further aspect, increasing the level of PGC-1α protein and/or PGC-1α gene in the subject is associated with decreased levels of PGC-1α protein and/or PGC-1α gene (e.g., neurological disease or condition). degenerative diseases and disorders). In some aspects, the present disclosure provides a method of increasing LRRK2 protein and/or LRRK2 gene levels in a subject in need thereof, comprising a miR-485 inhibitor. It relates to a method comprising administering to a subject. In a further aspect, increasing the level of LRRK2 protein and/or LRRK2 gene in a subject is used to treat diseases or conditions (e.g., neurodegenerative diseases and disorders) associated with decreased levels of LRRK2 protein and/or LRRK2 gene. can be useful. In some aspects, the disclosure provides a method of increasing NRG1 protein and/or NRG1 gene levels in a subject in need thereof, comprising a miR-485 inhibitor. It relates to a method comprising administering to a subject. In a further aspect, increasing NRG1 protein and/or NRG1 gene levels in a subject is used to treat diseases or conditions (e.g., neurodegenerative diseases and disorders) associated with decreased levels of NRG1 protein and/or NRG1 gene. can be useful. In some aspects, the present disclosure provides a method of increasing STMN2 protein and/or STMN2 gene levels in a subject in need thereof, comprising a miR-485 inhibitor. It relates to a method comprising administering to a subject. In a further aspect, increasing STMN2 protein and/or STMN2 gene levels in a subject is used to treat diseases or conditions (e.g., neurodegenerative diseases and disorders) associated with decreased STMN2 protein and/or STMN2 gene levels. can be useful. In some aspects, the disclosure provides a method of increasing VLDLR protein and/or VLDLR gene levels in a subject in need thereof, comprising a miR-485 inhibitor. It relates to a method comprising administering to a subject. In a further aspect, increasing VLDLR protein and/or VLDLR gene levels in a subject is used to treat diseases or conditions (e.g., neurodegenerative diseases and disorders) associated with decreased VLDLR protein and/or VLDLR gene levels. can be useful. In some aspects, the disclosure provides a method of increasing NRXN1 protein and/or NRXN1 gene levels in a subject in need of increasing NRXN1 protein and/or NRXN1 gene levels, comprising: It relates to a method comprising administering to a subject. In a further aspect, increasing NRXN1 protein and/or NRXN1 gene levels in a subject is used to treat diseases or conditions (e.g., neurodegenerative diseases and disorders) associated with decreased levels of NRXN1 protein and/or NRXN1 gene. can be useful. In some aspects, the disclosure provides a method of increasing GRIA4 protein and/or GRIA4 gene levels in a subject in need thereof, comprising a miR-485 inhibitor. It relates to a method comprising administering to a subject. In a further aspect, increasing levels of GRIA4 protein and/or GRIA4 gene in a subject is used to treat diseases or conditions (e.g., neurodegenerative diseases and disorders) associated with decreased levels of GRIA4 protein and/or GRIA4 gene. can be useful. In some aspects, the present disclosure provides a method of increasing NXPH1 protein and/or gene levels in a subject in need thereof, comprising a miR-485 inhibitor. It relates to a method comprising administering to a subject. In a further aspect, increasing NXPH1 protein and/or NXPH1 gene levels in a subject is used to treat diseases or conditions (e.g., neurodegenerative diseases and disorders) associated with decreased levels of NXPH1 protein and/or NXPH1 gene. can be useful. In some aspects, the present disclosure is a method of increasing PSD-95 protein and/or PSD-95 gene levels in a subject in need thereof. and methods comprising administering a miR-485 inhibitor to a subject. In a further aspect, increasing PSD-95 protein and/or PSD-95 gene levels in a subject is associated with decreased PSD-95 protein and/or PSD-95 gene levels (e.g., neurological disease or condition). degenerative diseases and disorders). In some aspects, the present disclosure provides a method of increasing synaptophysin protein and/or synaptophysin gene levels in a subject in need thereof, comprising a miR-485 inhibitor. It relates to a method comprising administering to a subject. In a further aspect, increasing the level of synaptophysin protein and/or synaptophysin gene in a subject is used to treat diseases or conditions (e.g., neurodegenerative diseases and disorders) associated with decreased levels of synaptophysin protein and/or synaptophysin gene. can be useful. In some aspects, the present disclosure provides a method of reducing caspase 3 protein and/or caspase 3 gene levels in a subject in need thereof, comprising miR- A method comprising administering a 485 inhibitor to a subject. In a further aspect, decreasing levels of caspase-3 protein and/or caspase-3 gene in a subject is associated with increased levels of caspase-3 protein and/or caspase-3 gene (e.g., neurodegenerative diseases and disorders). ) can be useful in the treatment of

Before describing this disclosure in more detail, it is to be understood that this disclosure is not limited to specific compositions or process steps, as the specific compositions or process steps described may, of course, vary. . Each of the individual aspects described and illustrated herein may be modified into several other aspects, without departing from the scope or spirit of the present disclosure, as will be apparent to those of ordinary skill in the art upon reading this disclosure. It has separate components and features that can be easily separated from or combined with any feature. Any method described can be performed in the order of events described or in any other order that is logically possible.

The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. Since the scope of the present disclosure is limited only by the appended claims, the terminology used herein is for the purpose of describing particular aspects only and not as limiting. It should also be understood that it is not intended to

I. Terminology Certain terms are first defined so that the present disclosure may be more readily understood. As used in this application, unless otherwise specified herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

An entity denoted by the term "a" or "an" refers to one or more of that entity, e.g., "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Note the point. Thus, "a" (or "an"), as well as "one or more," and "at least one," may be used interchangeably herein. . It is also noted that the claims may be drafted to exclude any optional element. Thus, this statement serves as an antecedent to the use of words of exclusion such as "only," "only," etc., in connection with the recitation of claim elements, or to the use of negative qualifications. shall be

Additionally, "and/or" when used herein is to be taken as specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and/or" as used herein in phrases such as "A and/or B" means "A and B", "A or B", "A" (alone), and " B" (alone) is intended to be included. Similarly, the term "and/or" when used in phrases such as "A, B, and/or C" is defined in the following aspects: A, B, and C; A, B, or C; or C; A or B; B or C; A and C; A and B; B and C;

Whenever an aspect is described herein with the term "comprising," it is described with the term "consisting of" and/or "consisting essentially of." It should be understood that other similar aspects are also provided.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. . See, eg, The Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed. , 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed. , 1999, Academic Press, and The Oxford Dictionary Of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide those skilled in the art with a general dictionary for many of the terms used in this disclosure.

Units, prefixes, and symbols are given in their International System of Units (SI)-approved form. Numeric ranges shall be inclusive of the numbers defining the range. When a numerical range is stated, each intervening integral value between the stated upper and lower limits of that range, and each fraction thereof, also includes each subrange between such values. It should be understood that the points specifically disclosed with. The upper and lower limits of any range may independently be included in the range or excluded from the range, with either limit being inclusive or neither limit being inclusive. Each range that includes either or both of the limits is also encompassed in the present disclosure. Accordingly, ranges recited herein are to be understood as shorthand for all values within that range, including the recited endpoints. For example, the range 1-10 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. be done.

It should be understood that where values are explicitly recited, values that are approximately the same number or amount as the recited value are also included within the scope of this disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is included within the scope of the disclosure. Conversely, where different elements or groups of elements are disclosed individually, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having multiple options, examples of that disclosure excluding each option alone or in any combination with other options are also disclosed herein. be done. That is, multiple elements of a given disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

Nucleotides are referred to by their accepted one-letter abbreviations. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Nucleotides are designated herein by the commonly known single letter symbols for nucleotides recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, "a" represents adenine, "c" represents cytosine, "g" represents guanine, "t" represents thymine, and "u" represents uracil.

Amino acid sequences are written left to right in the direction from amino terminus to carboxy terminus. Amino acids are referred to herein by their commonly known three-letter symbols or single-letter symbols for amino acids as recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The term "about" is used herein to mean about, approximately, approximately, or within. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify numerical values above and below the stated value, for example, with a variation of 10 percent (higher or lower) above or below.

As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B). , AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh. 74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (Virol. 33:375 (2004)), as well as any other AAV now known or later discovered (see, e.g., FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers)). In some aspects, "AAV" includes known derivatives of AAV. In some aspects, "AAV" includes modified or engineered AAV.

The terms "administration," "administering," and grammatical variations thereof refer to introducing a composition, such as a miRNA inhibitor of the present disclosure, to a subject by a pharmaceutically acceptable route. Introduction of compositions, such as micelles, comprising miRNA inhibitors of the present disclosure into a subject can be intratumoral, oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal , intralymphatic, intrathecal, periocular or topical. Administration includes self-administration and administration by another person. A suitable route of administration allows the composition or agent to perform its intended function. For example, where a suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject intravenously.

As used herein, the term "related to" refers to a close relationship between two or more entities or properties. For example, when used to describe a disease or condition treatable by the present disclosure (e.g., a disease or condition associated with abnormal levels of SIRT1 protein and/or SIRT1 gene), "associated with" The term means that a subject is likely to have the disease or condition if the subject exhibits aberrant expression of the protein and/or gene. In some aspects, abnormal protein and/or gene expression causes a disease or condition. In some embodiments, aberrant expression does not necessarily cause, but correlates with, a disease or condition. Non-limiting examples of suitable methods that can be used to determine whether a subject exhibits aberrant expression of a protein and/or gene associated with a disease or condition are provided elsewhere in this disclosure. show.

As used herein, the term "approximately" as applied to one or more values of interest refers to values that are comparable to the stated reference value. In certain aspects, the term "approximately" means 10% (above or below the number) in both directions of the stated reference value, unless stated otherwise or otherwise clear from the context. , 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less (100% of the possible values of such number ).

As used herein, the term "conserved" refers to nucleotides or amino acids of polynucleotide or polypeptide sequences, respectively, that are found invariantly at the same position in two or more sequences being compared. refers to residues. Relatively conserved nucleotides or amino acids are those that are more conserved between related sequences than the nucleotides or amino acids that occur in other portions of the sequences.

In some aspects, two or more sequences are said to be "completely conserved" or "identical" if they are 100% identical to each other. In some aspects, two or more sequences are " It is highly preserved." In some embodiments, the two or more sequences are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to each other is said to be "highly conserved". In some embodiments, two or more sequences are at least 30% identical to each other, at least 40% identical, at least 50% identical, at least 60% identical, or at least 70% identical to each other. It is said to be "conserved" if it is % identical, at least 80% identical, at least 90% identical, or at least 95% identical. In some aspects, two or more sequences are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical to each other, % identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical It is said that Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or can apply to portions, regions, or features thereof.

As used herein, the term "derived from" is isolated from or produced by using a particular molecule or organism or information (e.g., amino acid or nucleic acid sequence). It refers to a component that For example, a nucleic acid sequence derived from a second nucleic acid sequence can contain a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In the case of nucleotides or polypeptides, derived species can be obtained, for example, by naturally occurring mutagenesis, by artificial directed mutagenesis, or by artificial random mutagenesis. Mutagenesis used to derive nucleotides or polypeptides may be either intentionally directed or intentionally random, or a combination of each. Mutagenesis of nucleotides or polypeptides to create different nucleotides or polypeptides derived from the original can be a random event (e.g., caused by polymerase infidelity), resulting in Identification can be made, for example, by suitable screening methods described herein. In some aspects, the nucleotide or amino acid sequence derived from the second nucleotide or amino acid sequence is at least about 50%, at least about 51%, at least about 52%, at least about 53% relative to the second nucleotide or amino acid sequence, respectively. , at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63% , at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73% , at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83% , at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93% , at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity, wherein the first nucleotide Alternatively, the amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence.

As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide consisting of codons translatable into amino acids. A "stop codon" (TAG, TGA, or TAA) is not normally translated into amino acids but can be considered part of the coding region. However, all flanking sequences such as promoters, ribosome binding sites, transcription terminators, introns, etc. are not part of the coding region. The boundaries of the coding region are generally determined by a 5' start codon encoding the amino terminus of the resulting polypeptide and a 3' translation stop codon encoding the carboxy terminus of the resulting polypeptide.

The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each comprising a nucleobase sequence) that are related to each other by Watson-Crick base-pairing rules, or between an oligomer and a target gene. point to For example, the nucleobase sequence "TGA (5'→3')" is complementary to the nucleobase sequence "ACT (3'→5')". Complementarity may be "partial," in which less than all of the nucleobases of a given nucleobase sequence are matched to other nucleobases according to the base-pairing rules. For example, in some aspects, the complementarity between a given nucleobase sequence and another nucleobase sequence is about 70%, about 75%, about 80%, about 85%, about 90%, or about It can be 95%. Thus, in certain aspects, the term "complementarity" refers to at least about 80%, at least about 85%, at least about 90%, at least about 91%, Refers to a match or complementarity of at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Or as an example, "perfect" or "perfect" (100%) complementarity can exist between a given nucleobase sequence and another nucleobase sequence. In some aspects, the degree of complementarity between nucleobase sequences has significant effects on the efficiency and strength of hybridization between sequences.

The term "downstream" refers to a nucleotide sequence that is 3' to a reference nucleotide sequence. In certain aspects, the downstream nucleotide sequence relates to the sequence following the transcription start site. For example, the translation initiation codon of a gene is located downstream of the transcription initiation site.

The terms "excipient" and "carrier" are used interchangeably and refer to inert substances added to pharmaceutical compositions to further facilitate administration of a compound, eg, a miRNA inhibitor of the present disclosure.

As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product, such as RNA or polypeptide. Expression includes without limitation transcription of a polynucleotide into a microRNA binding site, small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product. Expression includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA) and translation of mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product may be a nucleic acid such as RNA produced by transcription of a gene. As used herein, a gene product may be a nucleic acid, RNA or miRNA produced by transcription of a gene, or a polypeptide translated from the transcript. Gene products described herein include nucleic acids with post-transcriptional modifications such as polyadenylation or splicing, or with phosphorylation, methylation, glycosylation, addition of lipids, association with other protein subunits, for example. , or polypeptides with post-translational modifications such as proteolytic cleavages.

As used herein, the term "homology" refers to the overall relatedness between polymer molecules, such as nucleic acid molecules. In general, the term "homology" refers to the evolutionary relationship between two molecules. Thus, two molecules that are homologous have a common evolutionary ancestry. In the context of this disclosure, the term homology encompasses both identity and similarity.

In some aspects, the polymer molecules are at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60% of the molecules. %, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the monomers are identical (exactly the same monomers) or are similar (conservative substitutions), they are considered to be "homologous" to each other. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide sequences).

In the context of this disclosure, substitutions (even when they are referred to as amino acid substitutions) are made at the nucleic acid level, i.e. replacing an amino acid residue with an alternative amino acid residue encodes the first amino acid. by replacing the codon that encodes the second amino acid with a codon that encodes the second amino acid.

As used herein, the term "identity" refers to the overall monomeric conservation between polymer molecules, eg, between polynucleotide molecules. The term "identical" without any additional modifiers, e.g., "polynucleotide A is identical to polynucleotide B", is 100% identical (100% sequence identity) between polynucleotide sequences means that For example, describing two sequences as "70% identical" is equivalent to describing them as having, eg, "70% sequence identity."

Calculation of percent identity of two polypeptide or polynucleotide sequences can be performed, for example, by aligning the two sequences for optimal comparison (e.g., primary and primary Gaps may be introduced in one or both of the two polypeptide or polynucleotide sequences and non-identical sequences may be ignored for comparison). In certain embodiments, the length of sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, At least 90%, at least 95%, or 100%. The amino acids, or bases in the case of polynucleotides, at corresponding amino acid positions are then compared.

When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. is a function of numbers. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

Suitable software programs that can be used to align different sequences (eg, polynucleotide sequences) are available from a variety of sources. One suitable program for determining percent sequence identity is one of the BLAST package programs available from the US Government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). There is a bl2seq that is part. Bl2seq performs comparisons between two sequences using either the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, part of the bioinformatics program package EMBOSS, available from the European Bioinformatics Institute (EBI) at the worldwide web. ebi. ac. Needle, Stretcher, Water or Matcher also available at uk/Tools/psa.

Sequence alignments can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE, and the like.

Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. Note that percent sequence identities are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded down to 80.18. Rounded up to 2. Also note that the length value is always an integer.

In certain aspects, the percentage identity (%ID) or the percentage identity (%ID) of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is %ID=100× (Y/Z), where Y is the amino acid residue ( or nucleobases) and Z is the total number of residues in the second sequence. The % identity of the first sequence to the second sequence is greater than the % identity of the second sequence to the first sequence if the length of the first sequence exceeds the length of the second sequence will be.

Those skilled in the art will appreciate that the generation of sequence alignments for calculating percent sequence identity is not limited to binary inter-sequence comparisons made solely with primary sequence data. Sequence alignments can be generated by combining sequence data with data from heterogeneous sources, such as structural data (e.g. protein crystal structures), functional data (e.g. positions of mutations), or phylogenetic data. It should also be understood. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is www.worldwideweb.com. t coffee. There is T-Coffee available at www.org or also available from eg EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity can be managed either automatically or manually.

As used herein, the terms "isolated," "purified," "extracted," and grammatical variations thereof are used interchangeably to refer to one or more purification processes. A desired composition of the present disclosure, eg, a preparation of a miRNA inhibitor of the present disclosure, that has been subjected to In some aspects, isolation or purification, as used herein, removes (e.g., fractions) a composition of the disclosure, e.g., a miRNA inhibitor of the disclosure, from a sample containing contaminants. It is the process of partial extraction.

In some embodiments, the isolated composition has no detectable undesired activity, or alternatively, the level or amount of the undesired activity is below an acceptable level or amount. In other aspects, the isolated composition has an acceptable amount and/or concentration and/or activity or greater amount and/or concentration of the desired composition of the present disclosure. In other aspects, the isolated composition is enriched compared to the starting material from which the composition is obtained. The enrichment is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999% , at least about 99.9999%, or greater than 99.9999%.

In some embodiments, an isolated preparation is substantially free of residual biological products. In some embodiments, the isolated preparation is 100% free of any contaminating biological material, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free. Residual biological products may include non-living substances (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.

As used herein, the term "linked" refers to a first amino acid sequence or polynucleotide sequence joined to a second amino acid sequence or polynucleotide sequence, respectively, by covalent or non-covalent bonds. . A first amino acid or polynucleotide sequence may be directly linked or juxtaposed to a second amino acid or polynucleotide sequence, or alternatively an intervening sequence may be covalently linked from the first sequence to the second sequence. can join. The term "ligated" refers to fusion of a first polynucleotide sequence to a second polynucleotide sequence at the 5' or 3' terminus, as well as to a second polynucleotide sequence (or a second polynucleotide sequence). It also includes insertions of the entire first polynucleotide sequence (or the second polynucleotide sequence, respectively) at any two nucleotides in the single polynucleotide sequence). A first polynucleotide sequence can be linked to a second polynucleotide sequence by a phosphodiester bond or linker. A linker can be, for example, a polynucleotide.

As used herein, "miRNA inhibitor" refers to a compound that can decrease, alter, and/or modulate miRNA expression, function, and/or activity. A miRNA inhibitor can be a polynucleotide sequence that is at least partially complementary to a target miRNA nucleic acid sequence, whereby the miRNA inhibitor hybridizes to the target miRNA sequence. For example, a miR-485 inhibitor of the present disclosure comprises a nucleotide sequence that encoded a nucleotide molecule that is at least partially complementary to a target miR-485 nucleic acid sequence, whereby the miR-485 inhibitor is a miR-485 sequence hybridize with. In a further aspect, hybridization of miR-485 to a miR-485 sequence reduces, alters, and/or modulates miR-485 expression, function, and/or activity (e.g., by hybridization, result in increased expression of SIRT1 protein and/or SIRT1 gene).

The terms "miRNA," "miR," and "microRNA" are used interchangeably and refer to microRNA molecules found in eukaryotes that are involved in gene regulation by RNA. This term is used to refer to a single-stranded RNA molecule that has been processed from a precursor. In some aspects, the term "antisense oligomer" can also be used to describe the microRNA molecules of the present disclosure. The names of miRNAs and their sequences relevant to this disclosure are provided herein. MicroRNAs recognize and bind to target mRNAs through imperfect base-pairing, resulting in destabilization or translational inhibition of target mRNAs, thereby down-regulating target gene expression. Conversely, targeting of miRNAs with molecules containing miRNA binding sites (generally molecules containing sequences complementary to the seed region of miRNAs) can reduce or inhibit miRNA-induced translational inhibition, leading to upregulation of target genes. bring.

The term “mismatch” or “multiple mismatches” refers to one or more mismatches that do not match a target nucleic acid sequence (eg, miR-485 inhibitor) according to the base-pairing rules in oligomeric nucleobase sequences (eg, miR-485 inhibitor). refers to the nucleobases (whether contiguous or spaced apart) of Although perfect complementarity is often desired, in some embodiments one or more (preferably six, five, four, three, two or one) to the target nucleic acid sequence. Mismatches can occur. Variations at any position within the oligomer are included. In certain aspects, the antisense oligomers (eg, miR-485 inhibitors) of the present disclosure comprise nucleobase sequence variations near the terminus, variations within the or within about 6, 5, 4, 3, 2, or 1 subunit of the 3' end. In some aspects, 1, 2, or 3 nucleobases may be removed and still provide on-target binding.

As used herein, the terms "modulate," "modify," and grammatical variations thereof generally when applied to a particular concentration, level, expression, function, or action, e.g. increasing or decreasing, e.g., directly or indirectly, promoting/stimulating/upregulating a particular concentration, level, expression, function, or behavior to act as an antagonist or agonist, or Refers to the ability to alter them by interfering with them/inhibiting them/down-regulating them. In some cases, the modulator increases a particular concentration, level, activity, or function relative to a control, or relative to a generally expected average level of activity, or relative to a control level of activity. may be increased and/or decreased. In some aspects, miRNA inhibitors disclosed herein, eg, miR-485 inhibitors, modulate (eg, decrease, alter, or eliminate) the expression, function and/or activity of miR-485 ), thereby modulating the expression and/or activity of the SIRT1 protein or gene.

"Nucleic acid," "nucleic acid molecule," "nucleotide sequence," "polynucleotide," and grammatical variations thereof are used interchangeably to refer to phosphate esters in either single-stranded form or double helix. A ribonucleoside (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleoside (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule") in polymeric form, or any phosphoester thereof Refers to analogs such as phosphorothioates and thioesters. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The terms nucleic acid molecule and in particular DNA or RNA molecule refer only to the primary and secondary structure of the molecule and are not limited to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (eg, restriction fragments), plasmids, supercoiled DNA, and chromosomes. In describing the structure of a particular double-stranded DNA molecule, sequence usually only provides sequence in the 5' to 3' direction along the non-transcribed strand of DNA (ie, the strand having sequence homology to mRNA). may be described herein according to the convention of A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the present disclosure comprises one or more nucleic acids as described herein.

The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", and grammatical variations thereof, are approved by regulatory agencies of the United States federal government for use in animals, including humans. any of the agents listed in the United States Pharmacopoeia or listed in the United States Pharmacopoeia, as well as inhibiting the biological activity and properties of the administered compound without causing undesirable physiological effects to the extent that administration of the composition to a subject is prohibited. any carrier or diluent that does not It includes desirable excipients and carriers that are useful in preparing pharmaceutical compositions and are generally safe and non-toxic.

As used herein, the term "pharmaceutical composition" means a compound or composition mixed or admixed with or in one or more other chemical ingredients, such as pharmaceutically acceptable carriers and excipients. Refers to one or more of the compounds described herein, eg, the miRNA inhibitors of the present disclosure, suspended in a. One purpose of a pharmaceutical composition is to facilitate administration of a formulation containing a miRNA inhibitor of the present disclosure to a subject.

As used herein, the term "polynucleotide" refers to a polymer of any length of nucleotides including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof.

In some embodiments, the term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double-, and single-stranded ribonucleic acid (“RNA”). The term also includes polynucleotides that have been modified, eg, by alkylation and/or capping, and unmodified forms of polynucleotides.

In some aspects, the term "polynucleotide" refers to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), spliced or unspliced tRNAs, rRNAs, shRNAs, siRNAs, Polyribonucleotides (containing D-ribose), any other type of polynucleotide that is N- or C-glycosides of purine or pyrimidine bases, including miRNA and mRNA, and others containing non-nucleotide backbones containing nucleobases in an arrangement that allows for base pairing and base stacking as found in polymers such as polyamides (e.g., peptide nucleic acid "PNA") and polymorpholino polymers, and in DNA and RNA. including other sequence-specific synthetic nucleic acid polymers that

In some aspects of the disclosure, a polynucleotide may be an oligonucleotide, such as, for example, an antisense oligonucleotide. In some aspects, the oligonucleotide is RNA. In some aspects, the RNA is synthetic RNA. In some aspects, the synthetic RNA includes at least one non-natural nucleobase. In some embodiments, all nucleobases of a particular type are replaced with non-natural nucleobases (e.g., all uridines in the polynucleotides disclosed herein are replaced with non-natural nucleobases, e.g. , 5-methoxyuridine).

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, eg, encoded by the SIRT1 gene. The polymer may contain modified amino acids. These terms may be modified naturally or through intervention such as any other manipulation or modification such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. Also included are modified amino acid polymers. For example, containing one or more amino acid analogs, including homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and unnatural amino acids such as creatine, and other modifications known in the art. Polypeptides are also included within the definition. As used herein, the term "polypeptide" refers to proteins, polypeptides, and peptides of any size, structure, or function.

Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.

A polypeptide can be a single polypeptide or can be a multimolecular complex such as a dimer, trimer, or tetramer. They can also include single-chain or multi-chain polypeptides. Most commonly, disulfide bonds are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of corresponding naturally occurring amino acids. In some aspects, a "peptide" is about 50 amino acids or less in length, e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35 amino acids. , about 40, about 45, or about 50 long.

As used herein, the terms "prevent," "preventing," and variations thereof refer to partially or completely delaying the onset of a disease, disorder, and/or condition; partially or completely delaying the onset of one or more symptoms, features or clinical signs of a disease, disorder and/or condition; one or more symptoms, features of a particular disease, disorder and/or condition , or partially or completely delay the onset of symptoms; partially or completely delay the progression of a particular disease, disorder, and/or condition; It refers to reducing the risk of developing pathologies that In some aspects, prevention of outcome is achieved by prophylactic treatment.

As used herein, the terms "promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, a coding sequence is positioned 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. Those skilled in the art will appreciate that different promoters can direct gene expression in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Promoters that direct a gene to be expressed in a particular cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters." Promoters that direct gene expression at specific stages of development or cell differentiation are commonly referred to as "development-specific promoters" or "cell differentiation-specific promoters." A promoter that is induced to express a gene following exposure or treatment of cells with an agent, biological molecule, chemical, ligand, light, etc. that induces the promoter is commonly referred to as an "inducible promoter" or a "regulatable promoter." called. It will further be appreciated that DNA fragments of different lengths may have the same promoter activity, since in many cases the exact boundaries of regulatory sequences have not been completely defined.

The boundaries of the promoter sequence are the transcription initiation site at its 3' end and the upstream (5' direction) so as to contain the minimum number of bases or elements required to initiate transcription at levels detectable above background. ). Within the promoter sequence will be found a transcription initiation site (conveniently defined for example by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. In some aspects, promoters that can be used with the present disclosure include tissue-specific promoters.

As used herein, "prevention" refers to therapeutic actions or courses of action used to prevent the onset of a disease or condition, or to prevent or delay symptoms associated with a disease or condition. Point.

As used herein, "prophylaxis" refers to measures taken to maintain health and prevent the onset of a disease or condition, or to prevent or delay symptoms associated with a disease or condition. .

As used herein, the terms "gene regulatory region" or "regulatory region" refer to upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of the coding region. ) and affects transcription, RNA processing, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, or stem-loop structures. When the coding region is for expression in eukaryotic cells, a polyadenylation signal and transcription termination sequence are commonly located 3' to the coding sequence.

In some aspects, miR-485 inhibitors (eg, polynucleotides encoding RNA comprising one or more miR-485 binding sites) disclosed herein are functionally linked to one or more coding regions. can include promoters and/or other expression (eg, transcription) control elements associated with In functional association, the coding region of a gene product is associated with one or more regulatory regions such that the expression of the gene product is under the influence or control of the regulatory region(s). For example, a coding region and a promoter are defined such that induction of promoter function results in transcription of the mRNA encoded by that coding region, and the nature of the linkage between the promoter and the coding region determines the ability of the promoter to direct expression of a gene product. are "functionally associated" if they do not interfere with the DNA template or the ability of the DNA template to be transcribed. Other expression control elements besides promoters, such as enhancers, operators, repressors, and transcription termination signals, can also be operatively associated with the coding region to direct expression of the gene product.

As used herein, the term "similarity" refers to the overall relatedness between polymer molecules, eg, polynucleotide molecules (eg, miRNA molecules). Calculations of % similarity between polymer molecules relative to each other are based on % identity, except that % similarity calculations take into account conservative substitutions as understood in the art. can be performed in the same manner as the calculation of Similarity (%) is a measure of comparison used, ie nucleic acids are compared, e.g., to their evolutionary closeness, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, or according to which combination thereof is compared.

The terms "subject," "patient," "individual," and "host," and variants thereof, are used interchangeably herein and include, but are not limited to, humans, domestic animals ( For diagnosis, treatment, or therapy, including in animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.). It refers to any desired mammalian subject, especially humans. The methods described herein are applicable to both human therapy and veterinary applications.

As used herein, the phrase "a subject in need thereof" refers to a miRNA inhibitor of the present disclosure (e.g., miR-485 inhibitory subject, such as a mammalian subject, that would benefit from administration of an agent).

As used herein, the term "therapeutically effective amount" means the desired therapeutic, pharmacological, and/or physiological effect in a subject necessary to produce the desired therapeutic, pharmacological, and/or physiological effect. Or an amount of a reagent or pharmaceutical compound, including a miRNA inhibitor of the present disclosure, sufficient to produce a physiological effect. A therapeutically effective amount can be a "prophylactically effective amount," where prevention can be considered therapy.

As used herein, the terms "treat," "treatment," or "treating" refer to, for example, reducing the severity of a disease or condition, reducing the duration of a disease course, a disease or condition (e.g. Refers to the amelioration or elimination of one or more symptoms associated with diabetes, diabetes), providing a beneficial effect to a subject with a disease or condition without necessarily treating the disease or condition. The term also includes prophylaxis or prevention of a disease or condition or symptoms thereof.

The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence.

"Vector" refers to any carrier for cloning and/or introducing nucleic acids into a host cell. A vector can be a replicon capable of joining another nucleic acid segment so as to effect replication of the joined segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous replicating unit in vivo (i.e., is capable of replicating under its own control). point to The term "vector" includes viral and non-viral carriers for the introduction of nucleic acids into cells in vitro, ex vivo, or in vivo. For example, numerous vectors are known and used in the art, including plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of the polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragment into the vector of choice with complementary cohesive termini.

Vectors can be engineered to encode selectable markers or reporters that allow for the selection or identification of cells that have taken up the vector. Expression of a selectable marker or reporter allows identification and/or selection of host cells which incorporate and express other coding regions contained in the vector. Examples of selectable markers known and used in the art include genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc., as well as genes used as phenotypic markers, That is, anthocyan regulatory genes, isopentenyltransferase genes and the like are included. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase ( Gus) and the like. Selectable markers can also be considered reporters.

II. Methods of Use In some aspects, the miR-485 inhibitors of the present disclosure modulate the expression and/or activity of one or more genes to exert therapeutic effects (e.g., in a subject afflicted with a neurodegenerative disease). can demonstrate. As described herein, in some aspects, the miR-485 inhibitors disclosed herein are SIRT1, CD36, PGC1, LRRK2, NRG1, STMN2, VLDLR, NRXN1, GRIA4, NXPH1, from DLG4 (also referred to herein as the "PSD-95 gene"), SYP (also referred to herein as the "synaptophysin gene"), CASP3 (also referred to herein as the "caspase-3 gene"), or combinations thereof The expression and/or activity of selected genes can be modulated. Without wishing to be bound by any one theory, such regulation may allow miR-485 inhibitors to regulate cellular homeostasis (e.g., CD36, SIRT1, PGC1α), including, but not limited to, , protein homeostasis (e.g., LRRK2 and SIRT1), autophagy-lysosomal pathway (e.g., SIRT1 and CD36), phagocytosis (e.g., CD36), glial cell biology (e.g., CD36 and SIRT1), neurogenesis/synaptogenesis (e.g., SIRT1, PGC1α, STMN2, and NRG1), neuroinflammation (e.g., CD36 and SIRT1), mitochondria-associated (e.g., PGC1α), and combinations thereof (e.g., SIRT1 and PGC1α) can affect many biological processes, including

Modulation of SIRT1 In some aspects, the present disclosure provides a method of increasing SIRT1 protein and/or SIRT1 gene expression in a subject in need thereof, comprising miR-485 Methods are provided that include administering to a subject a compound that inhibits activity (ie, a miR-485 inhibitor). In certain aspects, inhibiting miR-485 activity increases SIRT1 protein and/or SIRT1 gene expression in the subject.

Sirtuin 1 (SIRT1), also known as NAD-dependent deacetylase sirtuin 1, is a protein encoded by the SIRT1 gene in humans. The SIRT1 gene is located on human chromosome 10 (nucleotides 67,884,656 to 67,918,390 (+strand direction) of GenBank Accession No. NC_000010.11). Synonyms for the SIRT1 gene and its encoded protein are known, "regulatory protein SIR2 homolog 1", "silent mating-type information regulation2 homolog 1", "SIR2", "SIR2-like protein 1", "SIR2L1", "SIR2α", "type 1 sirtuin", "hSIRT1", or "hSIR2".

The human SIRT1 protein has at least two known isoforms resulting from alternative splicing. SIRT1 isoform 1 (UniProt Identifier: Q96EB6-1) consists of 747 amino acids and was selected as the canonical sequence (SEQ ID NO:31). SIRT1 isoform 2 (also known as "delta exon 8" (UniProt identification number: Q96EB6-2) consists of 561 amino acids and differs from the canonical sequence in the following ways: 454-639: deletion (SEQ ID NO: 32) Table 1 below shows the sequences of the two SIRT1 isoforms.

As used herein, the term "SIRT1" (including synonyms thereof) includes any variant or isoform of SIRT1 that is naturally expressed by the cell. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase SIRT1 isoform 1 expression. In some aspects, miR-485 inhibitors disclosed herein can increase SIRT1 isoform 2 expression. In a further aspect, miR-485 inhibitors disclosed herein can increase the expression of SIRT1 isoform 1 and isoform 2. Unless otherwise specified, both Isoform 1 and Isoform 2 are collectively referred to herein as "SIRT1."

In some embodiments, the miR-485 inhibitors of the present disclosure reduce SIRT1 protein and/or SIRT1 gene expression compared to a reference (eg, SIRT1 protein and/or SIRT1 gene expression in matched subjects who were not administered the miR-485 inhibitor). and/or reduce the expression of the SIRT1 gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Increase by 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, the miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, eg, miR-485-3p. by decreasing SIRT1 protein and/or SIRT1 gene expression. In some aspects, miR-485 inhibitors of the present disclosure can reduce the expression and/or activity of miR-485-3p.

In some embodiments, miR-485 inhibitors of the present disclosure reduce miR-485- 3p expression and/or activity by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Reduce by 80%, at least about 90%, or at least about 100%. In certain aspects, miR-485 inhibitors of the present disclosure reduce miR-485-5p relative to a reference (eg, expression of miR-485-5p in matched subjects not administered miR-485 inhibitor). expression and/or activity of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% %, at least about 90%, or at least about 100%. In a further aspect, the miR-485 inhibitors of the present disclosure are compared to a reference (eg, miR-485-3p and miR-485-5p expression in matched subjects who were not administered the miR-485 inhibitor) reducing the expression and/or activity of both miR-485-3p and miR-485-5p by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%; Reduce by at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. In some aspects, miR-485-3p and/or miR-485-5p expression is completely inhibited following administration of the miR-485 inhibitor.

As described herein, miR-485 inhibitors of the present disclosure can increase SIRT1 protein and/or SIRT1 gene expression when administered to a subject. Accordingly, in some aspects, the present disclosure provides SIRT1 protein and/or SIRT1 in a subject in need of treatment for a disease or condition associated with abnormal (e.g., decreased) levels of SIRT1 protein and/or SIRT1 gene. Methods of treating diseases or conditions associated with abnormal levels of genes are provided. In certain embodiments, the method comprises administering to the subject a compound that inhibits the activity of miR-485 (eg, a miR-485 inhibitor), wherein the miR-485 inhibitor increases levels of SIRT1 protein and/or SIRT1 gene. to increase

Modulation of CD36 As described herein, Applicants believe that the 3'UTR of human CD36 contains the target site for miR-485-3p and binding of miR-485-3p reduces CD36 expression. (see, eg, Examples 7 and 8). Accordingly, in some aspects, the present disclosure provides a method of increasing CD36 protein and/or CD36 gene expression in a subject in need thereof, comprising: miR-485 activity (ie, a miR-485 inhibitor) to a subject. In certain aspects, inhibiting miR-485 activity increases CD36 protein and/or CD36 gene expression in the subject.

Cluster determinant 36 (CD36), also known as platelet glycoprotein 4, is a protein encoded by the CD36 gene in humans. The CD36 gene is located on chromosome 7 (nucleotides 80,602,656 to 80,679,277 (+strand direction) of GenBank Accession No. NC_000007.14). Synonyms for the CD36 gene and its encoded protein are known, "platelet glycoprotein IV", "fatty acid transferase", "scavenger receptor class B member 3", "glycoprotein 88", "glycoprotein IIIb ", "Glycoprotein IV", "Thrombospondin Receptor", "GPIIIB", "PAS IV", "GP3B", "GPIV", "FAT", "GP4", "BDPLT10", "SCARB3", " "CHDS7", "PASIV", or "PAS-4".

The human CD36 protein has at least four known isoforms resulting from alternative splicing. CD36 isoform 1 (UniProt Identifier: P16671-1) consists of 472 amino acids and was selected as the canonical sequence (SEQ ID NO:36). CD36 isoform 2 (also known as "ex8-del" (UniProt identification number: P16671-2) consists of 288 amino acids and differs from the canonical sequence in the following ways: 274-288: SIYAVFESDVNLKGI→ETCVHFTSSFSVCKS, and 289-472: Deletion (SEQ ID NO: 37) CD36 isoform 3 (also known as "ex6-7-del" (UniProt Identifier: P16671-3) consists of 433 amino acids and is Differs from the canonical sequence: 234-272: Deletion (SEQ ID NO: 38) CD36 isoform 4 (also known as "ex4-del" (UniProt identification number: P16671-4) consists of 412 amino acids, the following 144-203: Deletion (SEQ ID NO: 39) Table 2 below shows the sequences of the four CD36 isoforms.

As used herein, the term "CD36" (including synonyms thereof) includes any variant or isoform of CD36 that is naturally expressed by the cell. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase CD36 isoform 1 expression. In some aspects, miR-485 inhibitors disclosed herein can increase CD36 isoform 2 expression. In some aspects, miR-485 inhibitors disclosed herein can increase CD36 isoform 3 expression. In some aspects, miR-485 inhibitors disclosed herein can increase CD36 isoform 4 expression. In a further aspect, the miR-485 inhibitors disclosed herein are CD36 isoforms 1 and 2, and/or isoforms 3 and 4, and/or isoforms 1 and 4, and /or the expression of both isoform 2 and isoform 3 can be increased. In some aspects, miR-485 inhibitors disclosed herein can increase expression of all CD36 isoforms. Unless otherwise specified, isoform 1, isoform 2, isoform 3, and isoform 4 are collectively referred to herein as "CD36."

In some embodiments, miR-485 inhibitors of the present disclosure reduce CD36 protein and/or CD36 gene expression compared to a reference (eg, CD36 protein and/or CD36 gene expression in a matched subject who was not administered a miR-485 inhibitor). and/or reduce the expression of the CD36 gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Increase by 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, the miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, thereby reducing CD36 Increase protein and/or CD36 gene expression. Two mature forms of miR-485-3p and miR-485-5p are known. As disclosed herein, in some aspects, miR-485 inhibitors of the present disclosure can reduce the expression and/or activity of miR-485-3p. In some aspects, miR-485 inhibitors can reduce the expression and/or activity of miR-485-5p. In a further aspect, miR-485 inhibitors disclosed herein can reduce the expression and/or activity of both miR-485-3p and miR-485-5p.

Modulation of PGC1 The disclosure provided herein provides that the miR-485 inhibitors of the disclosure further modulate the expression of PGC-1α in subjects afflicted, for example, with the diseases or disorders disclosed herein. (see, eg, Example 3). Accordingly, in some aspects, the present disclosure provides methods for increasing PGC-1α protein and/or PGC-1α gene expression in a subject in need of increased PGC-1α protein and/or PGC-1α gene expression. and comprising administering to the subject a compound that inhibits miR-485 activity (ie, a miR-485 inhibitor). In certain aspects, inhibiting miR-485 activity increases PGC-1α protein and/or PGC-1α gene expression in the subject.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), also known as PPARG coactivator 1 alpha or ligand action modulator 6, is the protein encoded by the PPARGC1A gene in humans. The PGC-1α gene is located on human chromosome 4 (nucleotides 23,792,021 to 24,472,905 (+strand direction) of GenBank Accession No. NC_000004.12). Synonyms for the PGC-1α gene and its encoded protein are known, such as "PPARGC1A", "LEM6", "PGC1", "PGC1A", "PGC-1v", "PPARGC1", "PGC1α", or and "PGC-1(α)".

The human PGC-1α protein has at least nine known isoforms resulting from alternative splicing. PGC-1α isoform 1 (UniProt Identifier: Q9UBK2-1) consists of 798 amino acids and is selected as the canonical sequence (SEQ ID NO:40). PGC-1α isoform 2 (also known as "isoform NT-7a" (UniProt identification number: Q9UBK2-2) consists of 271 amino acids and differs from the canonical sequence in the following ways: 269-271: DPK →LFL;272-798: Deletion (SEQ ID NO: 41) PGC-1α isoform 3 (also known as "isoform B5" (UniProt identification number: Q9UBK2-3) consists of 803 amino acids, the following Differs from the canonical sequence in: 1-18: MAWDMCNQDSESVWSDIE→MDETSPRLEEDWKKVLQREAGWQ (SEQ ID NO: 42) PGC-1α isoform 4 (also known as “isoform B4” (UniProt Identifier: Q9UBK2-4) has 786 and differs from the canonical sequence in the following ways: 1-18: MAWDMCNQDSESVWSDIE→MDEGYF (SEQ ID NO: 43) PGC-1α isoform 5 (also known as "isoform B4-8a" (UniProt identification number: Q9UBK2-5) consists of 289 amino acids and differs from the canonical sequence in the following ways: 1-18: MAWDMCNQDSESVWSDIE→MDEGYF, 294-301: LTPPTTPP→VKTNLISK, 302-798: deletion (SEQ ID NO:44). PGC-1α isoform 6 (also known as "isoform B5-NT" (UniProt identification number: Q9UBK2-6) consists of 276 amino acids and differs from the canonical sequence in the following ways: 1-18: MAWDMCNQDSESVWSDIE →MDETSPRLEEDWKKVLQREAGWQ, 269-271: DPK→LFL, 272-798: Deletion (SEQ ID NO: 45) PGC-1α isoform 7 (also known as "B4-3ext" (UniProt Identifier: Q9UBK2-7) is Consists of 138 amino acids and differs from the canonical sequence in the following ways: 1-18: MAWDMCNQDSESVWSDIE→MDEGYF, 144-150: LKKLLLA→VRTLPTV, 151-798: deletion (SEQ ID NO: 46) PGC-1α isoform 8. (Also known as "Isoform 8a" (UniProt Identifier: Q9UBK2-8) consists of 301 amino acids and differs from the canonical sequence in the following ways: 294-301: LTPPTTPP→VKTNLISK, 302-798: deletion Loss (SEQ ID NO: 47). PGC-1α isoform 9 (also known as "isoform 9" (UniProt identification number: Q9UBK2-9) consists of 671 amino acids and differs from the canonical sequence in the following ways: 1-127: deletion ( SEQ ID NO: 48).Table 3 below shows the sequences of the nine PGC-1α isoforms.

As used herein, the term "PGC-1α" (including synonyms thereof) includes any variant or isoform of PGC-1α that is naturally expressed by the cell. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase PGC-1α isoform 1 expression. In some aspects, miR-485 inhibitors disclosed herein can increase PGC-1α isoform 2 expression. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase PGC-1α isoform 1 expression. In some aspects, miR-485 inhibitors disclosed herein can increase PGC-1α isoform 2 expression. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase PGC-1α isoform 3 expression. In some aspects, miR-485 inhibitors disclosed herein can increase PGC-1α isoform 4 expression. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase PGC-1α isoform 5 expression. In some aspects, miR-485 inhibitors disclosed herein can increase PGC-1α isoform 6 expression. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase PGC-1α isoform 7 expression. In some aspects, miR-485 inhibitors disclosed herein can increase PGC-1α isoform 8 expression. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase PGC-1α isoform 9 expression. In a further aspect, the miR-485 inhibitors disclosed herein are PGC-1α isoform 1, isoform 2, isoform 3, isoform 4, isoform 5, isoform 6, isoform 7, isoform 7, Form 8 and isoform 9 expression can be increased. Unless otherwise specified, both isoform 1 and isoform 2 are collectively referred to herein as "PGC-1α."

In some embodiments, the miR-485 inhibitors of the present disclosure are compared to a reference (eg, PGC-1α protein and/or PGC-1α gene expression in matched subjects who were not administered the miR-485 inhibitor) to reduce the expression of PGC-1α protein and/or PGC-1α gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% , at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, the miR-485 inhibitors disclosed herein reduce miR-485 expression and/or activity, thereby reducing PGC activity. -1α protein and/or increase the expression of the PGC-1α gene. Two mature forms of miR-485 are known, miR-485-3p and miR-485-5p. In some aspects, miR-485 inhibitors of the present disclosure can reduce the expression and/or activity of miR-485-3p. In some aspects, miR-485 inhibitors can reduce the expression and/or activity of miR-485-5p. In a further aspect, miR-485 inhibitors disclosed herein can reduce the expression and/or activity of both miR-485-3p and miR-485-5p.

Modulation of LRRK2 In some aspects, the disclosure provided herein provides that the miR-485 inhibitors of the disclosure are effective in reducing the effects of the miR-485 inhibitors in subjects afflicted with the diseases or disorders (e.g., Parkinson's disease) disclosed herein. We demonstrate that the expression of LRRK2 can be regulated. Accordingly, in some aspects, the present disclosure provides a method of increasing LRRK2 protein and/or LRRK2 gene expression in a subject in need thereof, comprising: miR-485 activity (ie, a miR-485 inhibitor) to a subject. In certain aspects, inhibiting miR-485 activity increases LRRK2 protein and/or LRRK2 gene expression in the subject.

Leucine-rich repeat kinase 2 (LRRK2) is a kinase enzyme encoded by the LRRK2 gene in humans. The LRRK2 gene is located on human chromosome 12 (nucleotides 40,224,890 to 40,369,285 (+strand direction) of GenBank Accession No. NC_000012.12). Synonyms for the LRRK2 gene and its encoded protein are known and include PARK8, RIPK7, ROCO2, AURA17, and DARDARIN.

Table 4 below shows the amino acid sequence of the LRRK2 protein.

As used herein, the term "LRRK2" (including synonyms thereof) includes any variant or isoform of LRRK2 that is naturally expressed by the cell.

In some embodiments, the miR-485 inhibitors of the present disclosure reduce LRRK2 protein and/or LRRK2 gene expression compared to a reference (e.g., LRRK2 protein and/or LRRK2 gene expression in matched subjects who were not administered the miR-485 inhibitor). and/or reduce the expression of the LRRK2 gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Increase by 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, eg, miR-485-3p. to increase the expression of the LRRK2 protein and/or the LRRK2 gene.

Modulation of NRG1 The disclosure provided herein provides that miR-485 inhibitors of the present disclosure regulate NRG1 in subjects afflicted, for example, with the diseases or disorders disclosed herein (see, e.g., Parkinson's disease). Demonstrate that expression can be further regulated. Accordingly, in some aspects, the present disclosure provides a method of increasing NRG1 protein and/or NRG1 gene expression in a subject in need thereof, comprising: miR-485 activity (ie, a miR-485 inhibitor) to a subject. In certain aspects, inhibiting miR-485 activity increases NRG1 protein and/or NRG1 gene expression in the subject.

Neuregulin 1 is a cell adhesion molecule encoded by the NRG1 gene in humans. NRG1 is one of four proteins in the neuregulin family that act on the EGFR family of receptors. The NRG1 gene is located on human chromosome 8 (nucleotides 31,639,245 to 32,774,046 of GenBank Accession No. NC_000008.11). Synonyms for the NRG1 gene and its encoded protein are known and include "GGF", "HGL", "HRG", "NDF", "ARIA", "GGF2", "HRG1", "HRGA", " SMDF", "MST131", "MSTP131", and "NRG1-IT2".

The human NRG1 protein has at least 11 known isoforms resulting from alternative splicing. NRG1 isoform 1 (also known as 'α') (UniProt ID: Q02297-1) is 640 amino acids long and has been selected as the canonical sequence (SEQ ID NO:91). NRG1 isoform 2 (also known as "α1A") (UniProt Identifier: Q02297-2) is 648 amino acids long and differs from the canonical sequence in the following ways: 234-234: K→KHLGIEFIE ( SEQ ID NO:92). NRG1 isoform 3 (also known as "α2B") (UniProt Identifier: Q02297-3) is 462 amino acids long and differs from the canonical sequence in the following ways: (i) 424-462: YVSAMTTPAR . . . SPPVSSMTVS→HNLIAELRRRN. . . SSIPHLGFIL, and (ii) 463-640: deletion (SEQ ID NO:93). NRG1 isoform 4 (also known as "α3") (UniProt ID: Q02297-4) consists of 247 amino acids and differs from the canonical sequence in the following ways: (i) 234-247: KAEELYQKRVLTIT→SAQMSLLVIAAKTT , and (ii) 248-260: deletion (SEQ ID NO:94). NRG1 isoform 6 (also known as "β1" and "β1A") (UniProt Identifier: Q02297-6) is 645 amino acids long and differs from the canonical sequence in the following ways: 213-234: QPGFTGARCTENVPMKVQNQEK→PNEFTGDRCQNYVMASFYKHLGIEFME (SEQ ID NO: 95). NRG1 isoform 7 (also known as "β2" (UniProt ID: Q02297-7) consists of 647 amino acids and differs from the canonical sequence in the following ways: 213-233: QPGFTGARCTENVPMKVQNQE→PNEFTGDRCQNYVMASFY (SEQ ID NO: 96 NRG1 isoform 8 (also known as "β3" and "GGFHFB1") (UniProt Identifier: Q02297-8) consists of 241 amino acids and differs from the canonical sequence in the following ways: 213-241: QPGFTGARCTENVPMKVQNQEKAEELYQK→PNEFTGDRCQNYVMASFYSTSTPFLSLPE, and (ii) 242-640: deletion (SEQ ID NO: 97) NRG1 isoform 9 (also known as "GGF2" and "GGFHPP2") (UniProt Identifier: Q02297-9) has 422のアミノ酸からなり、以下の点でカノニカル配列と異なる：（ｉ）１～３３：ＭＳＥＲＫＥＧＲＧＫＧＫＧＫＫＫＥＲＧＳＧＫＫＰＥＳＡＡＧＳＱＳＰ→ＭＲＷＲＲＡＰＲＲＳ．．．ＥＶＳＲＶＬＣＫＲＣ、（２）１３４～１６８：ＥＩＩＴＧＭＰＡＳＴＥＧＡＹＶＳＳＥＳＰＩＲＩＳＶＳＴＥＧＡＮＴＳＳＳ→Ａ、（３）２１３～２４１：ＱＰＧＦＴＧＡＲＣＴＥＮＶＰＭＫＶＱＮＱＥＫＡＥＥＬＹＱＫ→ＰＮＥＦＴＧＤＲＣＱＮＹＶＭＡＳＦＹＳＴＳＴＰＦＬＳＬＰＥ , and (iv) 242-640: Deletion (SEQ ID NO: 98) NRG1 isoform 10 (also known as "SMDF") (UniProt Identifier: Q02297-10) consists of 296 amino acids and has the following Differs from the canonical sequence in: (i) 1-166: deletion, (ii) 167-167: S→MEIYSPDMSE...ETNLQTAPKL, (iii) 213-241: QPGFTGARCTENVPMKVQNQEKAEELYQK→PNEFTGDRCQNYVMASFYSTSTPFLSLPE, and (iv) 042 : deletion (SEQ ID NO: 99).NRG1 isoform 11 (also known as "Type IV-β1a") (UniProt Identifier: Q02297-11) is 590 amino acids long and canonical in that配列と異なる：（ｉ）１～２１：欠失、（ｉｉ）２２～３３：ＫＫＰＥＳＡＡＧＳＱＳＰ→ＭＧＫＧＲＡＧＲＶＧＴＴ、（ｉｉｉ）１３４～１６８：ＥＩＩＴＧＭＰＡＳＴＥＧＡＹＶＳＳＥＳＰＩＲＩＳＶＳＴＥＧＡＮＴＳＳＳ→Ａ、及び（ｉｖ）２１３～２３４：ＱＰＧＦＴＧＡＲＣＴＥＮＶＰＭＫＶＱＮＱＥＫ→ＰＮＥＦＴＧＤＲＣＱＮＹＶＭＡＳＦＹＫＨＬＧＩＥＦＭＥ（配列番号100). NRG1 isoform 12 (UniProt Identifier: Q02297-12) consists of 420 amino acids and differs from the canonical sequence in the following ways: (i) 213-233: QPGFTGARCTENVPMKVQNQE→PNEFTGDRCQNYVMASFY, and (ii) 424-640: deletion (SEQ ID NO: 101).

Table 5 below shows the amino acid sequences of NRG1 proteins, including known isoforms.

As used herein, the term "NRG1" (including synonyms thereof) includes any variant or isoform of NRG1 that is naturally expressed by the cell. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase expression of NRG1 isoform 1 (ie, canonical sequences). In some aspects, miR-485 inhibitors disclosed herein can increase NRG1 isoform 2 expression. In some aspects, miR-485 inhibitors disclosed herein can increase NRG1 isoform 3 expression. In some aspects, miR-485 inhibitors disclosed herein can increase NRG1 isoform 4 expression. In some aspects, miR-485 inhibitors disclosed herein can increase NRG1 isoform 6 expression. In some aspects, miR-485 inhibitors disclosed herein can increase NRG1 isoform 7 expression. In some aspects, miR-485 inhibitors disclosed herein can increase NRG1 isoform 8 expression. In some aspects, miR-485 inhibitors disclosed herein can increase NRG1 isoform 9 expression. In some aspects, miR-485 inhibitors disclosed herein can increase NRG1 isoform 10 expression. In some aspects, miR-485 inhibitors disclosed herein can increase NRG1 isoform 11 expression. In some aspects, miR-485 inhibitors disclosed herein can increase NRG1 isoform 12 expression. In a further aspect, the miR-485 inhibitors disclosed herein are NRG1 isoform 1, NRG1 isoform 2, NRG1 isoform 3, NRG1 isoform 4, NRG1 isoform 6, NRG1 isoform 7, NRG1 isoform Expression of form 8, NRG1 isoform 9, NRG1 isoform 10, NRG1 isoform 11, and NRG1 isoform 12 can be increased. Unless otherwise specified, the isoforms of NRG1 described above are collectively referred to herein as "NRG1."

In some embodiments, miR-485 inhibitors of the present disclosure reduce NRG1 protein and/or NRG1 gene expression compared to a reference (eg, NRG1 protein and/or NRG1 gene expression in a matched subject who was not administered a miR-485 inhibitor). and/or reduce the expression of the NRG1 gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Increase by 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, eg, miR-485-3p. to increase the expression of the NRG1 protein and/or the NRG1 gene.

Modulation of STMN2 The disclosure provided herein provides that the miR-485 inhibitors of the present disclosure modulate STMN2 in subjects afflicted, for example, with the diseases or disorders disclosed herein (see, eg, Parkinson's disease). Demonstrate that expression can be further regulated. Accordingly, in some aspects, the present disclosure provides a method of increasing STMN2 protein and/or STMN2 gene expression in a subject in need thereof, comprising: miR-485 activity (ie, a miR-485 inhibitor) to a subject. In certain aspects, inhibiting miR-485 activity increases STMN2 protein and/or STMN2 gene expression in the subject.

Stathmin-2 is a member of the stathmin family of phosphoproteins and is encoded in humans by the STMN2 gene. Stathmin proteins function in microtubule dynamics and signal transduction. The encoded protein has a regulatory role in neuronal growth and is thought to be involved in osteogenesis. The STMN2 gene is located on human chromosome 8 (nucleotides 79,611,117 to 79,666,162 of NC_000008.11). Synonyms for the STMN2 gene and its encoded protein are known and include "SCG10" and "SCGN10."

The human STMN2 protein has at least two known isoforms resulting from alternative splicing. STMN2 isoform 1 (UniProt Identifier: Q93045-1) is 179 amino acids long and has been selected as the canonical sequence (SEQ ID NO: 102). STMN2 isoform 2 (UniProt Identifier: Q93045-2) is 187 amino acids long and differs from the canonical sequence in the following ways: 161-179: ERHAAEVRRNKELQVELSG→LVKFISSELKESIESQFLELQREGEKQ (SEQ ID NO: 103).

Table 6 below shows the amino acid sequence of the STMN2 protein.

As used herein, the term "STMN2" (including synonyms thereof) includes any variant or isoform of STMN2 that is naturally expressed by the cell. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase expression of STMN2 isoform 1 (ie, canonical sequences). In some aspects, miR-485 inhibitors disclosed herein can increase STMN2 isoform 2 expression. In some aspects, miR-485 inhibitors disclosed herein can increase the expression of STMN2 isoform 1 and STMN2 isoform 2. Unless otherwise specified, the isoforms of STMN2 described above are collectively referred to herein as "STMN2."

In some embodiments, miR-485 inhibitors of the present disclosure reduce STMN2 protein and/or STMN2 gene expression compared to a reference (eg, STMN2 protein and/or STMN2 gene expression in a matched subject who was not administered a miR-485 inhibitor). and/or reduce the expression of the STMN2 gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Increase by 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, the miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, eg, miR-485-3p. by decreasing STMN2 protein and/or STMN2 gene expression.

Modulation of VLDLR The disclosure provided herein demonstrates that the miR-485 inhibitors of the present disclosure regulate VLDLR in subjects afflicted, for example, with the diseases or disorders disclosed herein (see, eg, Alzheimer's disease). Demonstrate that expression can be further regulated. Accordingly, in some aspects, the present disclosure provides a method of increasing VLDLR protein and/or VLDLR gene expression in a subject in need thereof, comprising: miR-485 activity (ie, a miR-485 inhibitor) to a subject. In certain aspects, inhibiting miR-485 activity increases VLDLR protein and/or VLDLR gene expression in the subject.

Very low density lipoprotein receptor (VLDLR) is a transmembrane lipoprotein receptor of the low density lipoprotein (LDL) receptor family. VLDLR is expressed in many tissues and plays an important role in various biological processes, including neuronal migration in the developing brain. In humans, VLDLR is encoded by the VLDLR gene located on chromosome 9 (nucleotides 2,621,786 to 2,660,056 of NC_000009.12). Synonyms for the VLDLR gene and its encoded protein are known and include "CAMRQ1", "CARMQ1", "CHRMQ1", "VLDLRCH" and "VLDL-R".

The human VLDLR protein has at least two known isoforms resulting from alternative splicing. The long VLDLR isoform (UniProt Identifier: P98155-1) is 873 amino acids long and was selected as the canonical sequence (SEQ ID NO: 111). The short VLDLR isoform (UniProt Identifier: P98155-2) is 845 amino acids long and differs from the canonical sequence in the following ways: 751-779: STATTVTYSETKDTNTTEISATSGLVPGG→R SEQ ID NO: 112). Table 7 below shows the amino acid sequence of the VLDLR protein.

As used herein, the term "VLDLR" (including synonyms thereof) includes any variant or isoform of VLDLR that is naturally expressed by the cell. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase expression of long VLDLR isoforms (ie, canonical sequences). In some aspects, miR-485 inhibitors disclosed herein can increase expression of short VLDLR isoforms. In some aspects, miR-485 inhibitors disclosed herein can increase expression of long and short VLDLR isoforms. Unless otherwise specified, the VLDLR isoforms described above are collectively referred to herein as "VLDLR."

In some embodiments, miR-485 inhibitors of the present disclosure reduce VLDLR protein and/or VLDLR gene expression in comparison to a reference (eg, VLDLR protein and/or VLDLR gene expression in a matched subject who was not administered a miR-485 inhibitor). and/or reduce the expression of the VLDLR gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Increase by 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, the miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, eg, miR-485-3p. to increase the expression of the VLDLR protein and/or the VLDLR gene.

Modulation of NRXN1 The disclosure provided herein provides that miR-485 inhibitors of the present disclosure regulate NRXN1 in subjects afflicted, for example, with the diseases or disorders disclosed herein (see, eg, Alzheimer's disease). Demonstrate that expression can be further regulated. Accordingly, in some aspects, the present disclosure provides a method of increasing NRXN1 protein and/or NRXN1 gene expression in a subject in need thereof, comprising: miR-485 activity (ie, a miR-485 inhibitor) to a subject. In certain aspects, inhibiting miR-485 activity increases NRXN1 protein and/or NRXN1 gene expression in the subject.

Neurexin 1 (NRXN1) is the protein encoded by the NRXN1 gene in humans. The NRXN1 gene is located on human chromosome 2 (nucleotides 49,918,503 to 51,032,536 of NC_000003.12). Synonyms for the NRXN1 gene and its encoded protein are known and include "PTHSL2", "SCZD17" and "Hs.22998".

The human NRXN1 protein has at least six known isoforms that arise from alternative promoter usage and alternative splicing. NRXN1 isoform 1a (UniProt Identifier: Q9ULB1-1) consists of 1,477 amino acids and was selected as the canonical sequence (SEQ ID NO: 104). NRXN1 isoform 2a (UniProt identifier: Q9ULB1-2) consists of 1,496 amino acids and differs from the isoform 1a canonical sequence in the following ways: 379-386: deletion, 1239-1239: A→AGNNDNERLAIARQRIPYRLGRVVDEWLLDK , 1373-1375: deletion (SEQ ID NO: 105). NRXN1 isoform 3a (UniProt identifier: Q9ULB1-3) consists of 1,547 amino acids and differs from the isoform 1a canonical sequence in the following ways: 258-258: → EIKFGLQCVLPVLLHDNDQGKYCCINTAKPLTEK, 386-386: M → MVNKLHCS 1239-1239: A→AGNNDNERLAIARQRIPYRLGRVVDEWLLDK (SEQ ID NO: 106). NRXN1-β isoform 4 (UniProt identifier: Q9ULB1-4) consists of 139 amino acids and differs from the isoform 1a canonical sequence in the following ways: 1-1335: deletion, 1336-1344: GKPPPTKEPI→MDMRWHCEN. ; 1373-1375: Deletion (SEQ ID NO: 107). NRXN1 isoform 1b (UniProt Identifier: P58400-2) consists of 472 amino acids and has been selected as the canonical sequence of NRXN1-β (SEQ ID NO: 108). NRXN1-β isoform 3b (UniProt Identifier: P58400-1) consists of 442 amino acids and differs from the isoform 1b canonical sequence in the following ways: 205-234: deletion (SEQ ID NO: 109).

Table 8 below shows the sequences of the six NRXN1 isoforms.

As used herein, the term "NRXN1" (including synonyms thereof) includes any variant or isoform of NRXN1 and NRXN1-β that is naturally expressed by the cell. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase expression of NRXN1 isoform 1a. In some aspects, miR-485 inhibitors disclosed herein can increase expression of NRXN1 isoform 2a. In some aspects, miR-485 inhibitors disclosed herein can increase expression of NRXN1 isoform 3a. In some aspects, miR-485 inhibitors disclosed herein can increase NRXN1 isoform 4 expression. In some aspects, miR-485 inhibitors disclosed herein can increase expression of NRXN1-β isoform 1b. In some aspects, miR-485 inhibitors disclosed herein can increase expression of NRXN1-β isoform 3b. In a further aspect, the miR-485 inhibitors disclosed herein are NRXN1 isoform 1a, NRXN1-β isoform 1b, NRXN1 isoform 2a, NRXN1 isoform 3a, NRXN1-β isoform 3b, and NRXN1 isoform 3b. Expression of one or more of Form 4 can be increased. Unless otherwise specified, NRXN1 isoform 1a, NRXN1-β isoform 1b, NRXN1 isoform 2a, NRXN1 isoform 3a, NRXN1-β isoform 3b, and NRXN1 isoform 4 are collectively referred to herein as "NRXN1". call.

In some embodiments, the miR-485 inhibitors of the present disclosure reduce NRXN1 protein and/or NRXN1 gene expression compared to a reference (eg, NRXN1 protein and/or NRXN1 gene expression in matched subjects who were not administered the miR-485 inhibitor). and/or reduce the expression of the NRXN1 gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Increase by 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, the miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, eg, miR-485-3p. by decreasing NRXN1 protein and/or NRXN1 gene expression.

Modulation of GRIA4 In some aspects, the miR-485 inhibitors of the present disclosure further modulate GRIA4 expression in subjects suffering from, for example, a disease or disorder disclosed herein (see, eg, Alzheimer's disease). can do. Accordingly, in some aspects, the present disclosure provides a method of increasing GRIA4 protein and/or GRIA4 gene expression in a subject in need thereof, comprising: miR-485 activity (ie, a miR-485 inhibitor) to a subject. In certain aspects, inhibiting miR-485 activity increases GRIA4 protein and/or GRIA4 gene expression in the subject.

Glutamate receptor 4 (GRIA4) is a member of the family of L-glutamate-gated ion channels that mediate fast synaptic excitatory neurotransmission. These channels are also responsive to the glutamate agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). In humans, GRIA4 is encoded by the GRIA4 gene located on chromosome 11 (nucleotides 105,609,540 to 105,982,092 of NC_000011.10). Synonyms for the GRIA4 gene and its encoded protein are known, "GLUR4", "GLURD", "GluA4", "GLUR4C", "NEDSGA", and "glutamate ionotropic receptor AMPA type subunit 4". ” is mentioned.

Human GRIA4 has at least two known isoforms that arise from alternative splicing. GRIA4 isoform 1 (UniProt Identifier: P48058-1) consists of 902 amino acids and was selected as the canonical sequence (SEQ ID NO: 113). GRIA4 isoform 2 (UniProt Identifier: P48058-2) is 433 amino acids long and differs from the canonical sequence in the following ways: (i) 424-433: ESPYVMYKKN→PLMKNPILRN, and (ii) 434- 902: Deletion (SEQ ID NO: 114).

Table 9 below shows the sequences of the different GRIA4 isoforms.

As used herein, the term "GRIA4" (including synonyms thereof) includes any variant or isoform of GRIA4 that is naturally expressed by the cell. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase expression of GRIA4 isoform 1 (ie, canonical sequences). In some aspects, miR-485 inhibitors disclosed herein can increase GRIA4 isoform 2 expression. In some aspects, miR-485 inhibitors disclosed herein can increase expression of GRIA4 isoform 1 and GRIA4 isoform 2. Unless otherwise specified, the above-described GRIA4 isoforms are collectively referred to herein as "GRIA4."

In some embodiments, the miR-485 inhibitors of the present disclosure reduce GRIA4 protein and/or GRIA4 gene expression compared to a reference (eg, GRIA4 protein and/or GRIA4 gene expression in a matched subject who was not administered the miR-485 inhibitor). and/or reduce the expression of the GRIA4 gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Increase by 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, the miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, eg, miR-485-3p. by decreasing the GRIA4 protein and/or GRIA4 gene expression.

Modulation of NXPH1 In some aspects, miR-485 inhibitors of the present disclosure further modulate expression of NXPH1 in subjects suffering from, for example, the diseases or disorders disclosed herein (see, eg, Alzheimer's disease). can do. Accordingly, in some aspects, the present disclosure provides a method of increasing NXPH1 protein and/or NXPH1 gene expression in a subject in need thereof, comprising: miR-485 activity (ie, a miR-485 inhibitor) to a subject. In certain aspects, inhibiting miR-485 activity increases NXPH1 protein and/or NXPH1 gene expression in the subject.

Neurexophilin-1 (NXPH1) is the protein encoded by the NXPH1 gene in humans. The NXPH1 gene is a member of the neurexophilin family and encodes a secreted protein with a variable N-terminal domain, a highly conserved N-glycosylated central domain, a short linker region, and a cysteine-rich C-terminal domain. there is This protein forms a very tight complex with alpha-neurexins, a group of proteins that promote adhesion between dendrites and axons. In humans, the NXPH1 gene is located on chromosome 7 (nucleotides 8,433,609 to 8,752,961 of NC_000007.14). Synonyms for the NXPH1 gene and its encoded protein are known and include "NPH1" and "Nbla00697".

Table 10 below shows the amino acid sequence of the NXPH1 protein.

As used herein, the term "NXPH1" (including synonyms thereof) includes any variant or isoform of NXPH1 that is naturally expressed by the cell.

In some embodiments, miR-485 inhibitors of the present disclosure reduce NXPH1 protein and/or NXPH1 gene expression compared to a reference (e.g., NXPH1 protein and/or NXPH1 gene expression in a matched subject who was not administered a miR-485 inhibitor). and/or reduce the expression of the NXPH1 gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Increase by 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, eg, miR-485-3p. to increase the expression of NXPH1 protein and/or NXPH1 gene.

Modulation of PSD-95 In some aspects, miR-485 inhibitors of the present disclosure reduce PSD-95 levels in subjects afflicted with, for example, the diseases or disorders disclosed herein (see, eg, Alzheimer's disease). Expression can be regulated. Accordingly, in some aspects, the present disclosure provides a method for enhancing expression of PSD-95 protein and/or PSD-95 gene (i.e., DLG4) in a subject in need of increased expression of PSD-95 protein and/or PSD-95 gene. A method of increasing expression is provided comprising administering to a subject a compound that inhibits miR-485 activity (ie, a miR-485 inhibitor). In certain aspects, inhibiting miR-485 activity increases PSD-95 protein and/or PSD-95 gene expression in the subject.

Postsynaptic density protein 95 (PSD-95), also known as synaptic associated protein 90 (SAP-90), is activated in humans by the DLG4 (disc large homolog 4) gene (also referred to herein as the "PSD-95 gene"). It is the encoded protein. The DLG4 gene is located on human chromosome 17 (nucleotides 7,187,180 to 7,220,050 (-strand direction) of GenBank Accession No. NC_000017.11). Synonyms for the DLG4 gene and its encoded protein are known and include "disc large MAGUK scaffold protein 4", "MRD62", "PSD95" and "SAP90".

The human PSD-95 protein has at least three known isoforms resulting from alternative splicing. PSD-95 isoform 1 (also known as PSD95-α) (UniProt Identifier: P78352-1) consists of 724 amino acids and has been selected as the canonical sequence (SEQ ID NO: 116). PSD-95 isoform 2, also known as PSD95-β (UniProt Identifier: P78352-2), consists of 767 amino acids and differs from the canonical sequence in the following ways: 1-10: MDCLCIVTTK→MSQRPRAPRSALWLLAPPLLRWAPPLLTVLHSDLFQALLDILDYYEASLSESQ (sequence No. 117) PSD-95 isoform 3 (UniProt Identifier: P78352-3) consists of 721 amino acids and differs from the canonical sequence in the following ways: 51-53: deletion (SEQ ID NO: 118).

Table 11 below shows the amino acid sequences of PSD-95 proteins, including known isoforms.

As used herein, the term "PSD-95" (including synonyms thereof) includes any variant or isoform of PSD-95 that is naturally expressed by the cell. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase PSD-95 isoform 1 expression. In some aspects, miR-485 inhibitors disclosed herein can increase PSD-95 isoform 2 expression. In some aspects, miR-485 inhibitors can increase PSD-95 isoform 3 expression. In a further aspect, miR-485 inhibitors disclosed herein can increase expression of PSD-95 isoform 1, PSD-95 isoform 2, and PSD-95 isoform 3. Unless otherwise specified, the isoforms of PSD-95 described above are collectively referred to herein as "PSD-95."

In some embodiments, the miR-485 inhibitors of the present disclosure are compared to a reference (eg, PSD-95 protein and/or PSD-95 gene expression in matched subjects who were not administered the miR-485 inhibitor) to reduce PSD-95 protein and/or PSD-95 gene expression by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% , at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, the miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, eg, miR-485-3p. to increase expression of PSD-95 protein and/or PSD-95 gene.

Modulation of Synaptophysin The disclosure provided herein is that the miR-485 inhibitors described herein suffer from, for example, the diseases or disorders disclosed herein (see, e.g., Parkinson's disease). It further demonstrates that the expression of synaptophysin in a subject can be modulated. Accordingly, in some aspects, the present disclosure provides a method of increasing expression of synaptophysin protein and/or synaptophysin gene (i.e., SYP) in a subject in need thereof. and administering to the subject a compound that inhibits miR-485 activity (ie, a miR-485 inhibitor). In certain aspects, inhibiting miR-485 activity increases synaptophysin protein and/or synaptophysin gene expression in the subject.

Synaptophysin, also known as the major synaptic vesicle protein p38, is the protein encoded in humans by the SYP gene (also referred to herein as the "synaptophysin gene"). The SYP gene is located on the short arm of the X chromosome (nucleotides 49,187,804 to 49,200,259 (-strand direction) of GenBank Accession No. NC_000023.11). Synonyms for the SYP gene and its encoded protein are known and include "MRX96" and "MRXSYP".

The synaptophysin protein has at least two known isoforms resulting from alternative splicing. Synaptophysin isoform 1 (UniProt Identifier: P08247-1) consists of 313 amino acids and is selected as the canonical sequence (SEQ ID NO: 119). Synaptophysin isoform 2 (UniProt Identifier: P08247-2) is 195 amino acids long and differs from the canonical sequence in the following ways: 1-118: deletion (SEQ ID NO: 120).

Table 12 below shows the amino acid sequences of synaptophysin proteins, including known isoforms.

As used herein, the term "synaptophysin" (including synonyms thereof) includes any variant or isoform of synaptophysin that is naturally expressed by the cell. Thus, in some aspects, miR-485 inhibitors disclosed herein can increase synaptophysin isoform 1 expression. In some aspects, miR-485 inhibitors disclosed herein can increase synaptophysin isoform 2 expression. In a further aspect, miR-485 inhibitors disclosed herein can increase the expression of both synaptophysin isoform 1 and synaptophysin isoform 2. Unless otherwise specified, the isoforms of synaptophysin described above are collectively referred to herein as "synaptophysin."

In some embodiments, miR-485 inhibitors of the present disclosure reduce synaptophysin protein and/or synaptophysin gene expression compared to a reference (e.g., expression of synaptophysin protein and/or synaptophysin gene in a matched subject who was not administered a miR-485 inhibitor). and/or reduce the expression of the synaptophysin gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Increase by 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300%.

Without being bound by any one theory, in some aspects, the miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, eg, miR-485-3p. by decreasing synaptophysin protein and/or synaptophysin gene expression.

Modulation of Caspase-3 In some aspects, the disclosure provided herein provides that miR-485 inhibitors of the disclosure afflicted with a disease or disorder (eg, Parkinson's disease) disclosed herein. Demonstrate that the expression of caspase-3 in a subject can be regulated. Accordingly, in some aspects, the present disclosure provides methods for reducing caspase-3 protein and/or caspase-3 gene (i.e., CASP3) expression in a subject in need thereof. A method of reducing expression is provided comprising administering to a subject a compound that inhibits miR-485 activity (ie, a miR-485 inhibitor). In certain aspects, inhibiting miR-485 activity reduces caspase-3 protein and/or caspase-3 gene expression in the subject.

Caspase-3 is a member of the cysteine-aspartic protease (caspase) family and plays a central role in cellular apoptosis by interacting with caspase-8 and caspase-9. In humans, the caspase-3 protein is encoded by the CASP3 gene (also referred to herein as the "caspase-3 gene"). The CASP3 gene is located on human chromosome 4 (nucleotides 184,627,696 to 184,649,509 (-strand direction) of GenBank Accession No. NC_000004.12). Synonyms for the CASP3 gene and its encoded protein are known and include "apopain", "CPP32", "SREBP cleaving activity 1", "protein yama", "SCA-1", "PARP cleaving protease", " pro-caspase 3”, and “Yama”.

Table 13 below shows the amino acid sequences of the caspase-3 protein precursors and the truncated forms of the caspase-3 proteins.

As used herein, the term “caspase-3” (including synonyms thereof) includes any variant or isoform of caspase-3 that is naturally expressed by the cell (eg, cleaved caspase-3).

In some embodiments, the miR-485 inhibitors of the present disclosure are compared to a reference (e.g., caspase-3 protein and/or caspase-3 gene expression in matched subjects who were not administered the miR-485 inhibitor) expression of caspase-3 protein and/or caspase-3 gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% %, at least about 80%, at least about 90%, or at least about 100%.

Without being bound by any one theory, in some aspects, the miR-485 inhibitors disclosed herein reduce the expression and/or activity of miR-485, eg, miR-485-3p. by decreasing the expression of the caspase 3 protein and/or the caspase 3 gene.

As will be apparent from the present disclosure, any disease or condition associated with abnormal (eg, decreased) levels of SIRT1 protein and/or SIRT1 gene can be treated according to the present disclosure. In some aspects, the present disclosure may be useful in treating diseases or conditions associated with abnormal (eg, decreased) levels of CD36 protein and/or CD36 gene. In some aspects, the present disclosure can also be used to treat diseases or disorders associated with abnormal (eg, decreased) levels of PGC-1α protein and/or PGC-1α gene. In some aspects, the present disclosure can also be used to treat diseases or disorders associated with abnormal (eg, decreased) levels of LRRK2 protein and/or LRRK2 gene. In some aspects, the present disclosure can also be used to treat diseases or disorders associated with abnormal (eg, decreased) levels of NRG1 protein and/or NRG1 gene. In some aspects, the present disclosure can also be used to treat diseases or disorders associated with abnormal (eg, decreased) levels of STMN2 protein and/or STMN2 gene. In some aspects, the present disclosure can also be used to treat diseases or disorders associated with abnormal (eg, decreased) levels of VLDLR proteins and/or VLDLR genes. In some aspects, the present disclosure can also be used to treat diseases or disorders associated with abnormal (eg, decreased) levels of NRXN1 protein and/or NRXN1 gene. In some aspects, the present disclosure can also be used to treat diseases or disorders associated with abnormal (eg, decreased) levels of GRIA4 protein and/or GRIA4 gene. In some aspects, the present disclosure can also be used to treat diseases or disorders associated with abnormal (eg, decreased) levels of NXPH1 protein and/or NXPH1 gene. In some aspects, the present disclosure can be used to treat diseases or disorders associated with abnormal (eg, decreased) levels of PSD-95 protein and/or PSD-95 gene. In some aspects, the present disclosure can be used to treat diseases or disorders associated with abnormal (eg, decreased) levels of synaptophysin protein and/or synaptophysin gene. In some aspects, the present disclosure can be used to treat diseases or disorders associated with abnormal (eg, increased) levels of caspase-3 protein and/or caspase-3 gene.

In some embodiments, diseases or conditions associated with abnormal (eg, decreased or increased) levels of such proteins and/or genes include neurodegenerative diseases or disorders. As used herein, the term "neurodegenerative disease or disorder" refers to a disease or disorder caused by progressive pathological changes in nerve cells of the nervous system, particularly the brain. In some aspects, such progressive destruction of the nervous system can result in physical (eg, ataxia) and/or mental (eg, dementia) impairment. Non-limiting examples of neurodegenerative diseases or disorders treatable by the present disclosure include Alzheimer's disease, Parkinson's disease, or any combination thereof. Other diseases or conditions treatable by the present disclosure include, but are not limited to, autism spectrum disorders, mental retardation, epileptic seizures, stroke, spinal cord injury, or any combination thereof. is mentioned.

In some aspects, diseases or disorders treatable by the present disclosure include Alzheimer's disease. In certain aspects, the Alzheimer's disease is predementia Alzheimer's disease, early Alzheimer's disease, moderate Alzheimer's disease, advanced Alzheimer's disease, early-onset familial Alzheimer's disease, inflammatory Alzheimer's disease, non-inflammatory Alzheimer's disease, cortical Alzheimer's disease, early onset Alzheimer's disease, late onset Alzheimer's disease, or any combination thereof.

In some embodiments, the disease or disorder treatable comprises Parkinsonism. As used herein, the term "parkinsonism" refers to a group of neurological disorders that cause the combination of movement abnormalities seen in Parkinson's disease. Non-limiting examples of such movement abnormalities include tremors, slow movements (bradykinesia), postural instability, loss of postural reflexes, flexed postures, freezing phenomena (feet temporarily “glued” to the ground). speech problems, muscle stiffness (stiffness), or a combination of these. In some aspects, the Parkinsonism is Parkinson's disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), normal pressure hydrocephalus (NSA), cerebrovascular Parkinsonism (also known as cerebrovascular disease), diffuse Lewy body disease, Parkinsonian dementia, X-linked dystonia Parkinsonism, secondary Parkinsonism (environmental etiologies such as toxins, drugs, postencephalitis, brain tumors, , head trauma, normal pressure hydrocephalus), or a combination thereof.

In some aspects, the parkinsonism treatable according to the present disclosure is Parkinson's disease. As used herein, the term "Parkinson's disease" (PD) results in motor and non-motor findings (i.e., symptoms) and is characterized by widespread degeneration of dopaminergic neurons in the nigro-striatal system. refers to a neurodegenerative disease that Non-limiting examples of motor and non-motor symptoms of PD are provided elsewhere in this disclosure. Proteinopathy (abnormal aggregation of α-synuclein) is a hallmark of PD. Other exemplary features of PD include dopaminergic neuronal damage, mitochondrial dysfunction, neuroinflammation, protein homeostasis (e.g., dysfunction in glial cells of autophagic clearance of damaged proteins and organelles). , and combinations thereof. While not wishing to be bound by any one theory, in certain aspects miR-485 inhibitors of the present disclosure can treat PD by ameliorating one or more of these characteristics of PD. .

In some aspects, administering a miR-485 inhibitor disclosed herein is one of the diseases or conditions associated with abnormal (e.g., decreased) levels of SIRT1 protein and/or SIRT1 gene The above symptoms can be improved. In some aspects, administering a miR-485 inhibitor disclosed herein is one of the diseases or conditions associated with abnormal (eg, decreased) levels of CD36 protein and/or CD36 gene. The above symptoms can be improved. In some aspects, administering a miR-485 inhibitor disclosed herein is associated with abnormal (eg, decreased) levels of PGC-1α protein and/or PGC-1α gene or One or more symptoms of the condition can be ameliorated. In some aspects, administering a miR-485 inhibitor disclosed herein is one of the diseases or conditions associated with abnormal (e.g., decreased) levels of LRRK2 protein and/or LRRK2 gene The above symptoms can be improved. In some aspects, administering a miR-485 inhibitor disclosed herein is one of the diseases or conditions associated with abnormal (e.g., decreased) levels of NRG1 protein and/or NRG1 gene The above symptoms can be improved. In some aspects, administering a miR-485 inhibitor disclosed herein is one of the diseases or conditions associated with abnormal (e.g., decreased) levels of STMN2 protein and/or STMN2 gene The above symptoms can be improved. In some aspects, administering a miR-485 inhibitor disclosed herein is one of the diseases or conditions associated with abnormal (e.g., decreased) levels of VLDLR protein and/or VLDLR gene The above symptoms can be improved. In some aspects, administering a miR-485 inhibitor disclosed herein is one of the diseases or conditions associated with abnormal (e.g., decreased) levels of NRXN1 protein and/or NRXN1 gene The above symptoms can be improved. In some aspects, administering a miR-485 inhibitor disclosed herein is one of the diseases or conditions associated with abnormal (e.g., decreased) levels of GRIA4 protein and/or GRIA4 gene The above symptoms can be improved. In some aspects, administering a miR-485 inhibitor disclosed herein is one of the diseases or conditions associated with abnormal (e.g., decreased) levels of NXPH1 protein and/or NXPH1 gene The above symptoms can be improved. In some aspects, administering a miR-485 inhibitor disclosed herein is associated with abnormal (eg, decreased) levels of PSD-95 protein and/or PSD-95 gene or One or more symptoms of the condition can be ameliorated. In some aspects, administering a miR-485 inhibitor disclosed herein is one of the diseases or conditions associated with abnormal (e.g., decreased) levels of synaptophysin protein and/or synaptophysin gene The above symptoms can be improved. In some aspects, administering a miR-485 inhibitor disclosed herein is associated with aberrant (eg, increased) levels of caspase-3 protein and/or caspase-3 gene or One or more symptoms of the condition can be ameliorated. Non-limiting examples of such symptoms are described below.

In some aspects, administering a miR-485 inhibitor of the present disclosure reduces the development or risk of developing one or more symptoms of cognitive impairment in a subject (eg, suffering from a neurodegenerative disease). , at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, compared to a reference (eg, a subject not administered a miR-485 inhibitor) %, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80 %, at least about 85%, at least about 90%, at least about 95%, or about 100%.

In some aspects, administering a miR-485 inhibitor of the present disclosure causes memory loss in a subject (eg, suffering from a neurodegenerative disease) to refer to (eg, memory loss in a subject prior to administration). decrease compared to In some aspects, administering a miR-485 inhibitor of the present disclosure reduces memory loss or the risk of developing amnesia in a subject (eg, suffering from a neurodegenerative disease). -485 inhibitor) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35% , at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% , at least about 90%, at least about 95%, or about 100%.

In some aspects, administering a miR-485 inhibitor of the present disclosure improves memory retention in a subject (eg, suffering from a neurodegenerative disease) by referring to (eg, memory retention in a subject prior to administration) improve compared to In some aspects, administering a miR-485 inhibitor of the present disclosure improves memory retention in a subject (eg, suffering from a neurodegenerative disease), see, eg, administering a miR-485 inhibitor. at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 40%, at least about about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least improved and/or increased by about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more.

In some aspects, administering a miR-485 inhibitor of the present disclosure reduces spatial working memory in a subject (eg, suffering from a neurodegenerative disease) to a reference (eg, spatial working memory in a subject prior to administration). improve compared to memory). As used herein, the term "spatial working memory" refers to the ability to sustain spatial information activity in working memory over short periods of time. In some aspects, spatial working memory is at least about 5%, at least about 10%, at least about 20%, at least about 30%, compared to a reference (eg, a subject not administered a miR-485 inhibitor) %, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250 %, or at least about 300% or more.

In some aspects, administering a miR-485 inhibitor of the present disclosure reduces the phagocytic activity of scavenger cells (eg, glial cells) in a subject (eg, suffering from a neurodegenerative disease) (eg, by increasing CD36 protein and/or CD36 gene expression), compared to a reference (eg, phagocytic activity in a subject prior to administration). In some aspects, administering a miR-485 inhibitor of the present disclosure reduces the density of neuronal dendritic spines in a subject (eg, suffering from a neurodegenerative disease) to a reference (eg, miR-485 485 inhibitor), at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, Increase by at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more.

In some aspects, administering a miR-485 inhibitor of the present disclosure reduces amyloid beta (Aβ) plaque burden (e.g., CD36 protein and/or (by increasing expression of the CD36 gene), reducing compared to a reference (eg, amyloid beta (Aβ) plaque burden in a subject prior to dosing). As used herein, "amyloid-beta plaques" refers to any form of abnormal deposit of amyloid-beta, including large aggregates and small aggregates of several amyloid-beta peptides, and any mutation of the amyloid-beta peptide. can include Amyloid-beta (Aβ) plaques are associated with, for example, abnormal synaptic composition, synaptic shape, synaptic density, loss of synaptic conductance, altered dendritic diameter, altered dendritic length, altered spine density, altered spine area. It is known to cause neuronal changes such as changes, changes in spine length, or changes in spine head diameter. In some aspects, administering a miR-485 inhibitor of the present disclosure reduces the amyloid-beta plaque burden in a subject (eg, suffering from a neurodegenerative disease) by referencing (eg, miR-485 inhibitor at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40% compared to non-treated subjects) , at least about 45%, at least about 50%, at least about 55%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.

In some aspects, administering a miR-485 inhibitor disclosed herein reduces neurogenesis (eg, CD36 protein and/or CD36 by increasing expression of the gene), compared to a reference (eg, neurogenesis in a subject prior to administration). As used herein, the term "neurogenesis" refers to the process by which nerve cells are formed. Neurogenesis involves the proliferation of neural stem and progenitor cells, the differentiation of these cells into new neural cell types, and the migration and survival of new cells. The term is intended to include neurogenesis, which occurs primarily during normal development of prenatal and perinatal development, as well as cell regeneration that occurs after disease, injury, or therapeutic intervention. Adult neurogenesis is also referred to as "neural" or "nervous" development. In some aspects, administering a miR-485 inhibitor of the present disclosure reduces neurogenesis in a subject (eg, suffering from a neurodegenerative disease) to a reference (eg, administering a miR-485 inhibitor). at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 70%, at least about Increase by about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or about 300% or more.

In some aspects, increasing and/or inducing neurogenesis is accompanied by increased proliferation, differentiation, migration, and/or survival of neural stem and/or progenitor cells. Thus, in some aspects, administering a miR-485 inhibitor of the present disclosure can increase proliferation of neural stem and/or progenitor cells in a subject. In certain aspects, neural stem and/or progenitor cell proliferation is at least about 5%, at least about 10%, at least about 20%, compared to a reference (eg, a subject not administered a miR-485 inhibitor) %, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200 %, at least about 250%, or at least about 300% or more. In some aspects, the neural stem and/or progenitor cell viability is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least Increase by about 200%, at least about 250%, or at least about 300% or more.

In some aspects, increasing and/or inducing neurogenesis involves increasing the number of neural stem and/or progenitor cells. In some aspects, the number of neural stem and/or progenitor cells is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about Increase by 200%, at least about 250%, or at least about 300% or more.

In some aspects, increasing and/or inducing neurogenesis involves increasing axonal, dendrite, and/or synaptic development. In certain aspects, axonal, dendrite, and/or synaptic development is at least about 5%, at least about 10%, compared to a reference (e.g., a subject not administered a miR-485 inhibitor), at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, Increase by at least about 200%, at least about 250%, or at least about 300% or more.

In some aspects, administering a miR-485 inhibitor disclosed herein prevents and/or inhibits the development of amyloid-beta plaque burden in a subject (eg, suffering from a neurodegenerative disease). do. In some aspects, administering a miR-485 inhibitor disclosed herein delays the onset of developing amyloid-beta plaque burden in a subject (eg, suffering from a neurodegenerative disease). In some aspects, administering a miR-485 inhibitor of the present disclosure reduces the risk of developing an amyloid-beta plaque burden in a subject (eg, suffering from a neurodegenerative disease).

In some aspects, administering a miR-485 inhibitor of the present disclosure increases neuronal dendritic spine density in a subject (eg, suffering from a neurodegenerative disease) by a reference (eg, prior to administration increase compared to neuronal dendritic spine density) in the subject. In some aspects, administering a miR-485 inhibitor of the present disclosure reduces dendritic spine density in a subject (eg, suffering from a neurodegenerative disease) by a reference (eg, miR-485 inhibitor at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40% %, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more.

In some aspects, administering a miR-485 inhibitor disclosed herein reduces loss of neuronal dendritic spines in a subject (e.g., suffering from a neurodegenerative disease), see (eg, loss of neuronal dendritic spines in the subject prior to administration). In some aspects, administering a miR-485 inhibitor reduces the loss of neuronal dendritic spines in a subject (eg, suffering from a neurodegenerative disease), see (eg, miR-485 inhibition) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about Reduce by 90%, at least about 95%, or about 100%.

In some aspects, administering a miR-485 inhibitor of the present disclosure reduces neuroinflammation (e.g., SIRT1 protein and/or SIRT1 gene expression) in a subject (e.g., suffering from a neurodegenerative disease). by increasing) to decrease compared to a reference (eg, neuroinflammation in a subject prior to administration). In certain aspects, administering a miR-485 inhibitor reduces neuroinflammation in a subject (eg, suffering from a neurodegenerative disease) to a reference (eg, a subject who has not been administered a miR-485 inhibitor). at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, compared to at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, Or reduce by about 100%. In some aspects, reducing neuroinflammation comprises reducing the amount of inflammatory mediators produced by glial cells. Thus, in certain aspects, administering a miR-485 inhibitor to a subject (eg, suffering from a neurodegenerative disease) reduces the amount of inflammatory mediators produced by glial cells to a reference (eg, miR-485 485 inhibitor), at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, Reduce by at least about 90%, at least about 95%, or by about 100%. In some aspects, the inflammatory mediator produced by glial cells comprises TNF-α. In some aspects, the inflammatory mediator comprises IL-1β. In some aspects, inflammatory mediators produced by glial cells include both TNF-α and IL-1β.

In some aspects, administering a miR-485 inhibitor disclosed herein reduces autophagy (eg, SIRT1 protein and/or SIRT1 increase (by increasing the expression of a gene). As used herein, the term "autophagy" refers to cellular stress responses and survival pathways responsible for the degradation of long-lived proteins, protein aggregates, and damaged organelles to maintain cellular homeostasis. . Not surprisingly, abnormalities in autophagy have been linked to numerous diseases, including many neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In some aspects, administering a miR-485 inhibitor of the present disclosure to a subject (eg, suffering from a neurodegenerative disease) is a reference (eg, a subject who has not been administered a miR-485 inhibitor) reduce autophagy by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about Increase by 95%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more. Increased autophagy can be measured by any suitable method known in the art. For example, in some aspects, increased autophagy can be observed by measuring the expression of genes associated with autophagosome formation (eg, LC3B).

In some aspects, administering a miR-485 inhibitor disclosed herein reduces α-secretase activity (e.g., SIRT1 protein and/or or by increasing expression of the SIRT1 gene). As used herein, the term "α-secretase" refers to a family of proteolytic enzymes that cleave amyloid precursor protein (APP) at its transmembrane region. Alpha-secretase is a member of the ADAM (“a disintegrin and metalloprotease domain”) family (eg, ADAM10) that is expressed on the surface of cells and anchored to the cell membrane. Specifically, α-secretase cleaves within a fragment that produces the Alzheimer's disease-associated peptide, amyloid beta, but APP is also processed by β-secretase and γ-secretase instead. Thus, in some aspects, α-secretase cleavage prevents the formation of amyloid beta and is considered part of the non-amyloidogenic pathway in APP processing. In some aspects, administering a miR-485 inhibitor disclosed herein to a subject (eg, suffering from a neurodegenerative disease) is referred to (eg, administering a miR-485 inhibitor) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 35%, at least about about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least Increase by about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more.

In some aspects, administering a miR-485 inhibitor disclosed herein reduces β-secretase 1 (BACE1) activity in a subject (eg, suffering from a neurodegenerative disease) (eg, by increasing the expression of the SIRT1 protein and/or the SIRT1 gene). As used herein, the term "β-secretase 1" or "BACE1" refers to an enzyme that is expressed primarily in neuronal cells. BACE1 is an aspartic protease important for the formation of the myelin sheath of peripheral nerve cells. In certain aspects, administering a miR-485 inhibitor disclosed herein to a subject (eg, suffering from a neurodegenerative disease) reduces BACE1 activity, see (eg, miR-485 inhibitor at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40% %, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, or about 100%.

As is well known in the art, many neurodegenerative diseases exhibit specific motor and/or non-motor symptoms. For example, non-limiting examples of motor symptoms associated with Parkinson's disease include resting tremor, decreased locomotor activity (bradykinesia), rigidity, postural instability, freezing feet, impaired handwriting (micrographia), Decreased facial expressions and uncontrollable jerky movements. Non-limiting examples of non-motor symptoms associated with Parkinson's disease include autonomic dysfunction, neuropsychiatric disorders (changes in mood, cognitive function, behavior, or thinking), sensory changes (particularly olfactory changes), and Difficulty sleeping.

In some aspects, administering a miR-485 inhibitor of the present disclosure reduces one or more motor symptoms in a subject (eg, suffering from a neurodegenerative disease) to a reference (eg, subject prior to administration) improvement compared to the corresponding motor symptoms in In some aspects, administering a miR-485 inhibitor of the present disclosure reduces one or more motor symptoms in a subject (eg, suffering from a neurodegenerative disease) to a reference (eg, miR-485 inhibition at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more.

In some aspects, administering a miR-485 inhibitor of the present disclosure reduces one or more motor symptoms in a subject (eg, suffering from a neurodegenerative disease) to a reference (eg, subject prior to administration) improve compared to one or more motor symptoms in In certain aspects, administering a miR-485 inhibitor disclosed herein reduces one or more non-motor symptoms in a subject (eg, suffering from a neurodegenerative disease), see (eg, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, compared to subjects who did not receive the miR-485 inhibitor) %, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% %, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more.

In some aspects, administering a miR-485 inhibitor disclosed herein reduces synaptic function in a subject (eg, suffering from a neurodegenerative disease) to a reference (eg, subject prior to administration). improve synaptic function in humans). As used herein, the term "synaptic function" refers to the ability of a synapse of a cell (eg, nerve cell) to transmit an electrical or chemical signal to another cell (eg, nerve cell). In some aspects, administering a miR-485 inhibitor of the present disclosure affects synaptic function in a subject (eg, suffering from a neurodegenerative disease) (eg, administering a miR-485 inhibitor). at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 40%, at least about about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least improve by about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more;

In some aspects, administering a miR-485 inhibitor of the present disclosure reduces loss of synaptic function in a subject (eg, suffering from a neurodegenerative disease) to a reference (eg, synaptic function in a subject prior to administration). can be prevented, delayed and/or ameliorated compared to loss of function). In some aspects, administering a miR-485 inhibitor reduces the loss of synaptic function in a subject (eg, suffering from a neurodegenerative disease) to a reference (eg, not administering a miR-485 inhibitor). at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%, prevents, delays, and/or ameliorates.

In some aspects, the miR-485 inhibitors disclosed herein can be administered by any suitable route known in the art. In certain aspects, the miR-485 inhibitor is administered supplementally, intramuscularly, subcutaneously, eye drops, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intracerebral It is administered intraventricularly, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, intratumorally, or any combination thereof. In certain aspects, the miR-485 inhibitor is administered intracerebroventricularly (ICV). In certain aspects, the miR-485 inhibitor is administered intravenously.

In some aspects, the miR-485 inhibitors of this disclosure can be used in combination with one or more additional therapeutic agents. In some aspects, the additional therapeutic agent and the miR-485 inhibitor are administered concurrently. In certain embodiments, the additional therapeutic agent and the miR-485 inhibitor are administered sequentially.

In some aspects, miR-485 inhibitors disclosed herein do not produce side effects. In certain aspects, miR-485 inhibitors of the disclosure do not adversely affect body weight when administered to a subject. In some aspects, the miR-485 inhibitors disclosed herein do not result in increased mortality or cause pathological abnormalities when administered to a subject.

III. miR-485 Inhibitors Useful in the Present Disclosure This disclosure discloses compounds capable of inhibiting miR-485 activity (miR-485 inhibitors). In some aspects, miR-485 inhibitors of the disclosure comprise a nucleotide sequence encoding a nucleotide molecule comprising at least one miR-485 binding site, wherein the nucleotide molecule does not encode a protein. As described herein, in some aspects, the miR-485 binding site is at least partially complementary to the target miRNA nucleic acid sequence (i.e., miR-485), thus miR-485 inhibitors hybridizes with nucleic acid sequences of miR-485.

In some aspects, the miR-485 binding sites of the miR inhibitors disclosed herein are at least about 50%, at least about 55%, at least about 60%, at least about 65% with the miR-485 nucleic acid sequence. %, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 %, or about 100% sequence identity. In certain aspects, the miR-485 binding site is fully complementary to the nucleic acid sequence of miR-485.

The hairpin precursor of miR-485 can generate both miR-485-5p and miR-485-3p. In the context of this disclosure, "miR-485" includes both miR-485-5p and miR-485-3p unless otherwise specified. Human mature miR-485-3p has the sequence 5'-GUCAUACACGGCUCUCCUCCUCU-3' (SEQ ID NO: 1; miRBase accession number MIMAT0002176). The 5' terminal sequence of miR-485-3p 5'-UCAUCA-3' (SEQ ID NO: 49) is the seed sequence. Human mature miR-485-5p has the sequence 5'-AGAGGCUGGCCGUGAUGAAUUC-3' (SEQ ID NO:33; miRBase accession number MIMAT0002175). The 5' terminal sequence of miR-485-5p 5'-GAGGCUG-3' (SEQ ID NO: 50) is the seed sequence.

As will be apparent to those skilled in the art, human mature miR-485-3p shares considerable sequence similarity with those of other species. For example, mouse mature miR-485-3p differs from human mature miR-485-3p by one amino acid at each of the 5′ and 3′ ends (ie, an extra “A” at the 5′ end). and no 'C' at the 3' end). Mouse mature miR-485-3p has the following sequence: 5′-AGUCAUACACGGCUCUCCUCUC-3′ (SEQ ID NO: 34; miRBase Accession No. MIMAT0003129; underlined corresponds to overlap with human mature miR-485-3p). . The sequence of mouse mature miR-485-5p is identical to the human sequence: 5'-agaggcuggccgugaugaauuc-3' (SEQ ID NO:33; miRBase accession number MIMAT0003128). Sequence alignments of human mature miR-485-3p and miR-485-5p with the corresponding sequences of other exemplary mammalian species are shown in FIGS. 5A and 5B. Because of such sequence similarities, in some embodiments, miR-485 inhibitors of the present disclosure are miR-485-3p and/or miR-485 inhibitors of one or more species shown in FIGS. 5A and 5B. -5p can bind. In certain aspects, miR-485 inhibitors disclosed herein can bind to human and mouse miR-485-3p and/or miR-485-5p.

In some aspects, the miR-485 binding site is a single-stranded polynucleotide sequence complementary (eg, fully complementary) to the sequence of miR-485-3p (or a subsequence thereof). In some aspects, the subsequence of miR-485-3p comprises a seed sequence. Thus, in certain aspects, the miR-485 binding site is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, the nucleic acid sequence set forth in SEQ ID NO:49, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% have sequence complementarity; In a particular aspect, the miR-485 binding site is complementary to miR-485-3p with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In a further aspect, the miR-485 binding site is fully complementary to the nucleic acid sequence set forth in SEQ ID NO:1.

In some aspects, the miR-485 binding site is a single-stranded polynucleotide sequence complementary (eg, fully complementary) to the sequence (or subsequence thereof) of miR-485-5p. In some aspects, the subsequence of miR-485-5p comprises a seed sequence. In certain aspects, the miR-485 binding site is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence complementarity have sex. In a particular aspect, the miR-485 binding site is complementary to miR-485-5p with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In a further aspect, the miR-485 binding site is fully complementary to the nucleic acid sequence set forth in SEQ ID NO:35.

Seed regions of miRNAs form tight duplexes with target mRNAs. Many miRNAs base-pair imperfectly with the 3' untranslated regions (UTRs) of target mRNAs, with the 5' proximal 'seed' region of the miRNA providing most of the pairing specificity. Without wishing to be bound by any theory, the first 9 miRNA nucleotides (including the seed sequence) confer higher specificity, whereas miRNA nucleotides 3' of this region are less It confers sequence specificity, thereby allowing a higher degree of mismatched base-pairing, with positions 2-7 thought to be the most important. Thus, in certain aspects of the disclosure, the miR-485 binding site comprises a sequence that is fully complementary (ie, 100% complementary) over the entire length of the miR-485 seed sequence.

The miRNA sequences and miRNA binding sequences that can be used in the context of the present disclosure include, but are not limited to, all or part of the sequences of the sequence listings provided herein, as well as miRNA precursors. sequences, or complements of one or more of these miRNAs. the sequence is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 71%, at least about 72% the mature sequence of the indicated miRNA or its complementary sequence , at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82% , at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92% , at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to or complementary to Any aspect of the present disclosure that includes specific miRNAs or miRNA binding sites by name to include sequences is also contemplated.

In certain aspects, the miRNA-binding sequences of the present disclosure are 5', 3' of the sequences shown in the sequence listing provided herein, as long as the modified sequence is still capable of specifically binding to miR-485. Additional nucleotides may be included at the ends, or at both the 5' and 3' ends. In some aspects, the miRNA binding sequences of the present disclosure are at least 1, 2, 3 relative to the sequences shown in the sequence listing provided, as long as the modified sequence is still capable of specifically binding miR-485. , 4, 5, 6, 7, 8, 9, 10, or more nucleotides.

It is also specifically contemplated that any methods and compositions described herein with respect to miRNA binding molecules or miRNAs can be practiced with synthetic miRNA binding molecules. It will also be appreciated that the disclosure relating to RNA sequences of this disclosure is equally applicable to the corresponding DNA sequences.

In some aspects, the miR-485 inhibitors of this disclosure have at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides at the 5' end of the nucleotide sequence. nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides , at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides. In some aspects, the miR-485 inhibitor has at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides at the 3' end of the nucleotide sequence; at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides.

In some aspects, the miR-485 inhibitors disclosed herein are from about 6 to about 30 nucleotides in length. In certain aspects, the miR-485 inhibitors disclosed herein are 7 nucleotides in length. In a further aspect, the miR-485 inhibitors disclosed herein are 8 nucleotides in length. In some aspects, the miR-485 inhibitor is 9 nucleotides in length. In some aspects, the miR-485 inhibitors of this disclosure are 10 nucleotides in length. In a particular aspect, the miR-485 inhibitor is 11 nucleotides in length. In a further aspect, the miR-485 inhibitor is 12 nucleotides in length. In some aspects, the miR-485 inhibitors disclosed herein are 13 nucleotides in length. In certain aspects, the miR-485 inhibitors disclosed herein are 14 nucleotides in length. In some aspects, the miR-485 inhibitors disclosed herein are 15 nucleotides in length. In a further aspect, the miR-485 inhibitor is 16 nucleotides in length. In certain aspects, the miR-485 inhibitors of this disclosure are 17 nucleotides in length. In some aspects, the miR-485 inhibitor is 18 nucleotides in length. In some aspects, the miR-485 inhibitor is 19 nucleotides in length. In certain aspects, the miR-485 inhibitor is 20 nucleotides in length. In a further aspect, the miR-485 inhibitors of this disclosure are 21 nucleotides in length. In some aspects, the miR-485 inhibitor is 22 nucleotides in length.

In some aspects, the miR-485 inhibitors disclosed herein are at least about 50%, at least about 55%, at least about 60%, at least about 65% with a sequence selected from SEQ ID NOS: 2-30. %, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 %, or about 100% identical nucleotide sequences. In certain aspects, the miR-485 inhibitor comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:2-30, wherein the nucleotide sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, or may optionally contain 10 mismatches.

In some aspects, the miRNA inhibitor is 5′-UGUAUGA-3′ (SEQ ID NO:2), 5′-GUGUAUGA-3′ (SEQ ID NO:3), 5′-CGUGUAUGA-3′ (SEQ ID NO:4), 5′-CCGUGUAUGA-3′ (SEQ ID NO: 5), 5′-GCGUGUAUGA-3′ (SEQ ID NO: 6), 5′-AGCCGUGUAUGA-3′ (SEQ ID NO: 7), 5′-GAGCCGUGAUGA-3′ (SEQ ID NO: 8 ), 5′-AGAGCCGUGUAUGA-3′ (SEQ ID NO: 9), 5′-GAGAGCGUGUAUGA-3′ (SEQ ID NO: 10), 5′-GGAGAGCCGUGUAUGA-3′ (SEQ ID NO: 11), 5′-AGGAGAGCCGGUGUAUGA-3′ (sequence No. 12), 5′-GAGGAGAGCCGUGUAUGA-3′ (SEQ ID NO: 13), 5′-AGAGGAGAGCCGGUGUAUGA-3′ (SEQ ID NO: 14), or 5′-GAGAGGAGAGCGUGUAUGA-3′ (SEQ ID NO: 15).

In some aspects, the miRNA inhibitor is 5′-UGUAUGAC-3′ (SEQ ID NO: 16), 5′-GUGUAUGAC-3′ (SEQ ID NO: 17), 5′-CGUGUAUGAC-3′ (SEQ ID NO: 18), 5'-CCGUGUAUUGAC-3' (SEQ ID NO: 19), 5'-GCGUGUAUUGAC-3' (SEQ ID NO: 20), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 21), 5'-GAGCCGUGUAUGAC-3' (SEQ ID NO: 22 ), 5′-AGAGCCGUGUAUGAC-3′ (SEQ ID NO: 23), 5′-GAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 24), 5′-GGAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 25), 5′-AGGAGAGCCGUGUAUGAC-3′ (sequence No. 26), 5′-GAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 27), 5′-AGAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 28), 5′-GAGAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 29), or AGAGAGGAGAGCCGUGUAUGAC (SEQ ID NO: 30) have

In some aspects, the miRNA inhibitor is 5′-TGTATGA-3′ (SEQ ID NO:62), 5′-GTGTATGA-3′ (SEQ ID NO:63), 5′-CGTGTATGA-3′ (SEQ ID NO:64), 5′-CCGTGTATGA-3′ (SEQ ID NO: 65), 5′-GCCGTGTATGA-3′ (SEQ ID NO: 66), 5′-AGCCGTGTATGA-3′ (SEQ ID NO: 67), 5′-GAGCCGTGTATGA-3′ (SEQ ID NO: 68 ), 5′-AGAGCCGTGTATGA-3′ (SEQ ID NO: 69), 5′-GAGAGCCGTGTATGA-3′ (SEQ ID NO: 70), 5′-GGAGAGCCGTGTATGA-3′ (SEQ ID NO: 71), 5′-AGGAGAGCCGTGTATGA-3′ (sequence No. 72), 5′-GAGGAGAGCCGTGTATGA-3′ (SEQ ID NO: 73), 5′-AGAGGAGAGCCGTGTATGA-3′ (SEQ ID NO: 74), 5′-GAGAGGAGAGCCGTGTATGA-3′ (SEQ ID NO: 75), 5′-TGTATGAC-3′ (SEQ ID NO: 76), 5′-GTGTATGAC-3′ (SEQ ID NO: 77), 5′-CGTGTATGAC-3′ (SEQ ID NO: 78), 5′-CCGTGTATGAC-3′ (SEQ ID NO: 79), 5′-GCCGTGTATGAC- 3′ (SEQ ID NO: 80), 5′-AGCCGTGTATGAC-3′ (SEQ ID NO: 81), 5′-GAGCCGTGTATGAC-3′ (SEQ ID NO: 82), 5′-AGAGCCGTGTATGAC-3′ (SEQ ID NO: 83), 5′- GAGAGCCGTGTATGAC-3′ (SEQ ID NO: 84), 5′-GGAGAGCCGTGTATGAC-3′ (SEQ ID NO: 85), 5′-AGGAGAGCCGTGTATGAC-3′ (SEQ ID NO: 86), 5′-GAGGAGAGCCGTGTATGAC-3′ (SEQ ID NO: 87), 5 '-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 88), 5'-GAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 89), and AGAGAGGAGAGCCGTGTATGAC (SEQ ID NO: 90).

In some aspects, the miRNA inhibitors (i.e., miR-485 inhibitors) disclosed herein are 5′-AGAGGAGAGCGUGUAUGAC-3′ (SEQ ID NO: 28) or 5′-AGAGGAGAGCCGTGTATGAC-3′ (SEQ ID NO: 28) 88) and at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least Contains nucleotide sequences that are about 95% identical. In some embodiments, the miRNA inhibitor comprises a nucleotide sequence having at least 90% similarity to 5'-AGAGGAGAGCCGGUAUUGAC-3' (SEQ ID NO:28) or 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:88). In some aspects, the miRNA inhibitor has a nucleotide sequence 5′-AGAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO:28) or 5′-AGAGGAGAGCCGTGTATGAC-3′ (SEQ ID NO:88) with one substitution or two substitutions. include. In some aspects, the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:28) or 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:88). In a particular aspect, the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:28).

In some aspects, the miR-485 inhibitors of the present disclosure comprise any one of the sequences disclosed herein, e.g., SEQ ID NOs: 2-30, and at least one, at least two, at least 3, at least 4, or at least 5 additional nucleic acids, at least 1, at least 2, at least 3, at least 4, or at least 5 additional nucleic acids at the C-terminus, or both. In some aspects, miR-485 inhibitors of the present disclosure comprise a sequence disclosed herein, e.g., any one of SEQ ID NOS: 2-30, and an additional nucleic acid at the N-terminus and/or C and one additional nucleic acid at the end. In some aspects, the miR-485 inhibitors of the present disclosure comprise any one of the sequences disclosed herein, e.g. and/or one or two additional nucleic acids at the C-terminus. In some aspects, the miR-485 inhibitors of the present disclosure comprise any one of the sequences disclosed herein, e.g. or 1-3 additional nucleic acids at the C-terminus. In some aspects, the miR-485 inhibitor comprises 5'-GAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:29).

In some aspects, the miR-485 inhibitors of this disclosure comprise one miR-485 binding site. In a further aspect, miR-485 inhibitors disclosed herein comprise at least two miR-485 binding sites. In certain aspects, the miR-485 inhibitor comprises three miR-485 binding sites. In some aspects, the miR-485 inhibitor comprises four miR-485 binding sites. In some aspects, the miR-485 inhibitor comprises 5 miR-485 binding sites. In certain aspects, the miR-485 inhibitor comprises 6 or more miR-485 binding sites. In some aspects, all miR-485 binding sites are the same. In some aspects, all miR-485 binding sites are different. In some aspects, at least one miR-485 binding site is different. In some aspects, all miR-485 binding sites are miR-485-3p binding sites. In another aspect, all miR-485 binding sites are miR-485-5p binding sites. In a further aspect, the miR-485 inhibitor comprises at least one miR-485-3p binding site and at least one miR-485-5p binding site.

III. a. Chemically Modified Polynucleotides In some aspects, miR-485 inhibitors disclosed herein comprise polynucleotides comprising at least one chemically modified nucleoside and/or nucleotide. When a polynucleotide of the present disclosure has been chemically modified, it can be referred to as a "modified polynucleotide."

A "nucleoside" is a compound containing a sugar molecule (e.g., pentose or ribose) or derivative thereof, together with an organic base (e.g., purine or pyrimidine) or derivative thereof (also referred to herein as a "nucleobase"). Point. "Nucleotide" refers to a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method, such as chemically, enzymatically, or recombinantly, to include one or more modified non-natural nucleosides.

A polynucleotide can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. Linkage can be a standard phosphodiester bond, in which case the polynucleotide comprises a region of nucleotides.

Modified polynucleotides disclosed herein can contain a variety of different modifications. In some aspects, the modified polynucleotide contains one, two, or more (optionally different) nucleoside or nucleotide modifications. In some aspects, the modified polynucleotide has one or more desired properties, e.g., improved thermal or chemical stability, reduced immunogenicity, reduced It may exhibit degradation, increased binding to target microRNAs, reduced non-specific binding to other microRNAs or other molecules.

In some aspects, the polynucleotides (eg, miR-485 inhibitors) of this disclosure are chemically modified. As used herein, the term "chemically modified" or, where appropriate, "chemically modified" with respect to polynucleotides includes adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribonucleosides thereof, including but not limited to their nucleobases, sugars, backbones, or any combination thereof, their positions, patterns, percentages, or populations refers to modifications in one or more of

In some aspects, the polynucleotides (eg, miR-485 inhibitors) of the present disclosure are uniformly chemically modified in all or any of the same nucleoside types, or of the same starting modification in all or any of the same nucleoside types. Groups of modifications generated by downward titration, or may have measured percent chemical modifications of all or any of the same nucleoside type but with random incorporation. In a further aspect, the polynucleotides (eg, miR-485 inhibitors) of the present disclosure can have 2, 3, or 4 uniform chemical modifications of the same nucleoside type throughout the polynucleotide (eg, All uridines and all cytosines, etc. are similarly modified).

Modified nucleotide base pairing can occur between standard adenine-thymine, adenine-uracil, or guanine-cytosine base pairs as well as between nucleotides and/or modified nucleotides, including non-standard or modified bases. base pairs wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between a non-canonical base and a canonical base or between two complementary non-canonical base structures to One example of such non-canonical base pairing is base pairing between the modified nucleobases inosine and adenine, cytosine, or uracil. Any combination of bases/sugars or linkers can be incorporated into the polynucleotides of the present disclosure.

Those skilled in the art will appreciate that unless otherwise noted, the polynucleotide sequences described in this application list a "T" in a representative DNA sequence, but when the sequence represents RNA, the "T" is replaced by a "U". will be understood to be replaced with For example, the TDs of this disclosure can be administered as RNA, as DNA, or as hybrid molecules containing both RNA and DNA units.

In some aspects, the polynucleotides (eg, miR-485 inhibitors) are at least 2 (eg, 2, 3, 4, 5, 6, 7, 8, 8, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 18, 20 or more) modified nucleobase combinations.

In some aspects, the nucleobases, sugars, backbone linkages, or any combination thereof in the polynucleotide are at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least about 30% , at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80% , at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% modified.

(i) Base Modifications In certain aspects, chemical modifications are present at the nucleobases within the polynucleotides of the present disclosure (eg, miR-485 inhibitors). In some aspects, at least one chemically modified nucleoside is a modified uridine (eg, pseudouridine (ψ), 2-thiouridine (s2U), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), or 5-methoxy-uridine (mo5U)), modified cytosine (e.g. 5-methyl-cytidine (m5C)), modified adenosine (e.g. 1-methyl-adenosine (m1A), N6- methyl-adenosine (m6A), or 2-methyladenine (m2A)), modified guanosines (eg, 7-methyl-guanosine (m7G) or 1-methyl-guanosine (m1G)), or combinations thereof.

In some aspects, the polynucleotides (eg, miR-485 inhibitors) of this disclosure are uniformly modified (eg, fully modified, modified throughout the sequence) for a particular modification. For example, polynucleotides can be uniformly modified with the same type of base modification, eg, 5-methyl-cytidine (m5C), which means that all cytosine residues in the polynucleotide sequence are replaced with 5-methyl-cytidine (m5C). ) is meant to be replaced with Similarly, a polynucleotide may be uniformly modified by replacement of any kind of nucleoside residue present in the sequence with a modified nucleoside, eg, any of those described above.

In some aspects, the polynucleotides (eg, miR-485 inhibitors) of the present disclosure comprise at least two (eg, two, three, four, or more) combinations of modified nucleobases. include. In some aspects, at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% are modified nucleobases .

(ii) Backbone Modifications In some aspects, the polynucleotides (ie, miR-485 inhibitors) of the present disclosure can include any useful internucleoside linkages. Such linkages, including backbone modifications, useful in the compositions of the present disclosure include, but are not limited to: 3′-alkylene phosphonates, 3′-aminophosphoroamido dart, alkene-containing backbone, aminoalkylphosphoramidate, aminoalkylphosphotriester, boranophosphate, _-CH2 -O-N( _CH3 ) _-CH2- , _-CH2 -N( _CH3 )-N (CH ₃ )—CH ₂ —, —CH ₂ —NH—CH ₂ —, chiral phosphonates, chiral phosphorothionates, formacetyl and thioformacetyl backbones, methylene (methylimino), methyleneformacetyl and thioformacetyl backbones, Methyleneimino and methylenehydrazino backbones, morpholino linkages, —N(CH ₃ )—CH ₂ —CH ₂ —, oligonucleosides with heteroatom internucleoside linkages, phosphinates, phosphoramidates, phosphorodithioates, phosphorothioate internucleosides Bonds, phosphorothionates, phosphotriesters, PNAs, siloxane backbones, sulfamate backbones, sulfide, sulfoxide and sulfone backbones, sulfonate and sulfonamide backbones, thionoalkylphosphonates, thionoalkylphosphotriesters, and thionophosphoro Amidate.

In some aspects, the presence of the backbone linkages disclosed above increases the stability and resistance to degradation of polynucleotides (eg, miR-485 inhibitors) of the disclosure.

In some aspects, at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least about 30%, at least about 35% of the polynucleotides (eg, miR-485 inhibitors) of the present disclosure %, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% %, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the backbone bonds are modified (e.g., all of them are phosphorothioate is).

In some aspects, backbone modifications that can be included in polynucleotides (ie, miR-485 inhibitors) of the present disclosure include phosphorodiamidite morpholino oligomers (PMO) and/or phosphorothioate (PS) modifications.

(iii) Sugar Modifications Modified nucleosides and nucleotides that can be incorporated into polynucleotides (eg, miR-485 inhibitors) of the present disclosure can be modified to the sugar of the nucleic acid. In some aspects, the sugar modification increases the affinity of binding of the miR-485 inhibitor to the miR-485 nucleic acid sequence. By incorporating affinity-improving nucleotide analogues, such as LNAs or 2'-substituted sugars, into miR-485 inhibitors, the length and/or size of miR-485 inhibitors can be reduced.

In some aspects, at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least about 30%, at least about 35% of the polynucleotides (i.e., miR-485 inhibitors) of the present disclosure %, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% %, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the nucleotides contain a sugar modification (eg, LNA).

In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotide units are sugar modified (eg, LNA).

Generally, RNA contains the sugar group ribose, which is a five-membered ring with oxygen. Non-limiting exemplary modified nucleotides include substitution of oxygen at ribose (e.g., with S, Se, or alkylene, e.g., methylene or ethylene); addition of double bonds (e.g., cyclopentenyl or cyclohexenyl of ribose) ring contraction of ribose (e.g. formation of a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g. with additional carbon or heteroatoms to form a 6- or 7-membered ring, e.g. morpholinos that also have anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and phosphoramidate backbones); polycyclic forms (e.g., tricyclo); and "unlocked" forms such as glycol nucleic acid ( GNA) (e.g., R-GNA or S-GNA, where the ribose is replaced with a glycol unit attached to the phosphodiester bond), threose nucleic acids (TNA, where the ribose is α-L-threofuranosyl- (3′→2′)), and peptide nucleic acids (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbones). A sugar group may also contain one or more carbons that have the opposite stereochemical configuration to the corresponding carbon in ribose. Thus, a polynucleotide molecule may comprise nucleotides containing, for example, arabinose as a sugar.

The 2' hydroxy group (OH) of ribose can be modified or substituted with a number of different substituents. Exemplary substitutions at the 2′-position include, but are not limited to, H, halo, optionally substituted C _1-6 alkyl; optionally substituted C _1-6 alkoxy; optionally substituted C _6-10 aryloxy; optionally substituted C _3-8 cycloalkyl; optionally substituted C _3-8 cycloalkoxy; optionally substituted C _6-10 aryloxy; optionally substituted C _6-10 aryl-C _1-6 alkoxy, optionally substituted C _1-12 (heterocyclyl)oxy; sugar (e.g. ribose, pentose, or any described herein); polyethylene glycol (PEG), —O( _CH2CH2O ) _nCH2CH2OR , where R is H or optionally _substituted alkyl _; , n is 0-20 (for example, 0-4, 0-8, 0-10, 0-16, 1-4, 1-8, 1-10, 1-16, 1-20, 2-4, 2-8, 2-10, 2-16, 2-20, 4-8, 4-10, 4-16, and 4-20)); "locked" nucleic acid (LNA) (2'- The hydroxy is linked to the 4′-carbon of the same ribose sugar by a C _1-6 alkylene or C _1-6 heteroalkylene bridge, where exemplary bridges include methylene, propylene, ether, amino bridges , aminoalkyl, aminoalkoxy, amino, and amino acids).

In some aspects, the nucleotide analogues present in the polynucleotides (ie, miR-485 inhibitors) of the present disclosure are, for example, 2′-O-alkyl-RNA units, 2′-OMe-RNA units, 2′ —O-alkyl-SNA, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, arabinonucleic acid (ANA) unit, 2′-fluoro-ANA unit, HNA unit, INA (intercalating nucleic acid ) units, 2′ MOE units, or any combination thereof. In some aspects, the LNA is, for example, an oxy LNA (eg, β-D-oxy-LNA or α-L-oxy-LNA), an amino LNA (eg, β-D-amino-LNA or α-L- amino-LNA), thio-LNA (eg, β-D-thio-LNA or α-L-thio-LNA), ENA (eg, β-D-ENA or α-L-ENA), or any combination thereof is. In a further aspect, nucleotide analogues that can be included in the polynucleotides (ie, miR-485 inhibitors) of the present disclosure include locked nucleic acids (LNA), unlocked nucleic acids (UNA), arabinonucleic acids (ABA), bridged nucleic acids (BNA) , and/or peptide nucleic acids (PNAs).

In some aspects, polynucleotides (ie, miR-485 inhibitors) of the present disclosure may comprise both modified RNA nucleotide analogs (eg, LNA) and DNA units. In some aspects, the miRNA inhibitor is a gapmer. For example, U.S. Pat. Nos. 8,404,649, 8,580,756, 8,163,708, 9,034,837 (all of which are incorporated herein by reference in their entireties). (incorporated herein). In some aspects, the miRNA inhibitor is a micromer. See US Patent Publication No. US20180201928, which is incorporated herein by reference in its entirety.

In some aspects, polynucleotides of the present disclosure (at least miR-485 inhibitors) can include modifications to prevent rapid degradation by endonucleases and exonucleases. Modifications include, but are not limited to, (a) terminal modifications such as 5′ terminal modifications (phosphorylation, dephosphorylation, conjugation, inverted binding, etc.), 3′ terminal modifications (conjugation (b) base modifications, such as modified bases, stabilized bases, destabilized bases, or substitutions with bases that base pair with a broad repertoire of partners, or conjugated bases; (c) sugar modifications (eg, at the 2' or 4' positions) or sugar substitutions, and (d) internucleoside linkage modifications, including modifications or substitutions of phosphodiester linkages.

IV. Vectors and Delivery Systems In some aspects, miR-485 inhibitors of the present disclosure are administered to subjects afflicted with diseases or conditions associated with aberrant (e.g., decreased) levels of, e.g., SIRT1 protein and/or SIRT1 gene. can be administered using any relevant delivery system known in the art. In certain aspects, the delivery system is a vector. Accordingly, in some aspects, the disclosure provides vectors comprising the miR-485 inhibitors of the disclosure.

In some aspects, the vector is a viral vector. In some aspects, the viral vector is an adenoviral vector or an adeno-associated viral vector. In some aspects, the viral vector is AAV with a serotype of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or any combination thereof. In some aspects, the adenoviral vector is a third generation adenoviral vector. ADEASY™ is by far the most common method for making adenoviral vector constructs. This system consists of two types of plasmids: shuttle (or transfer) vectors and adenoviral vectors. The transgene of interest is cloned into the shuttle vector, verified and linearized with the restriction enzyme PmeI. This construct was then transfected into BJ5183E. Transform ADEASIER-1 cells, which are E. coli cells. PADEASY™ is an approximately 33 Kb adenoviral plasmid containing the adenoviral genes necessary for virus production. The shuttle vector and adenoviral plasmid have matched left and right homology arms that facilitate homologous recombination of the transgene into the adenoviral plasmid. Standard BJ5183 can also be co-transformed with supercoiled PADEASY™ and shuttle vectors, but this method results in a high background of non-recombinant adenoviral plasmids. The recombinant adenoviral plasmid is then verified for size and proper restriction digestion pattern to confirm that the transgene has been inserted into the adenoviral plasmid and that no other pattern of recombination has occurred. Once verified, the recombinant plasmid is linearized with PacI to generate an ITR-flanked linear dsDNA construct. When 293 or 911 cells are transfected with the linearized constructs, virus can be harvested after approximately 7-10 days. In addition to this method, methods for making adenoviral vector constructs known in the art at the time this application was filed can be used to practice the methods disclosed herein.

In some aspects, the viral vector is a retroviral vector, such as a lentiviral vector (eg, a third or fourth generation lentiviral vector). Lentiviral vectors are usually produced in a transient transfection system in which one cell line is transfected with three separate plasmid expression systems. These include transfer vector plasmids (part of the HIV provirus), packaging plasmids or constructs, and plasmids containing the heterologous envelope genes (env) of different viruses. After placing the three plasmid components of the vector into the packaging cell, it is inserted into the HIV shell. Since the viral portion of the vector contains the insert sequence, the virus cannot replicate in the cell line. Current third-generation lentiviral vectors encode only three of the nine HIV-1 proteins (Gag, Pol, Rev), which prevent recombination from producing replication-competent virus. are expressed from separate plasmids for In 4th generation lentiviral vectors, the retroviral genome is further reduced (see, eg, TAKARA® LENTI-X™ 4th generation packaging system).

Any AAV vector known in the art can be used in the methods disclosed herein. AAV vectors may include known vectors and may include variants, fragments, or fusions thereof. In some aspects, the AAV vector is AAV type 1 (AAV1), AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh. 74, avian AAV, bovine AAV, canine AAV, equine AAV, goat AVV, primate AAV, non-primate AAV, bovine AAV, shrimp AVV, snake AVV, and any combination thereof.

In some aspects, the AAV vector is AAV1, AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh. 74, an AAV vector selected from the group consisting of avian AAV, bovine AAV, canine AAV, equine AAV, goat AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, and any combination thereof derived from

In some aspects, the AAV vector is AAV1, AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh. 74, avian AAV, bovine AAV, canine AAV, equine AAV, goat AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, and any combination thereof It is a chimeric vector derived from two AAV vectors.

In certain aspects, the AAV vector comprises at least two different AAV vector regions that are well known in the art.

In some aspects, the AAV vector comprises a first AAV (e.g., AAV1, AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh.74, AAV, bovine AAV, canine AAV, equine AAV, goat AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, or any combination thereof); of AAV (e.g., AAV1, AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, goat a second terminal inverted sequence derived from AAV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, or any combination thereof.

In some aspects, the AAV vector is AAV1, AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh. 74, an AAV vector selected from the group consisting of avian AAV, bovine AAV, canine AAV, equine AAV, goat AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, and any combination thereof including the part of In some aspects, the AAV vector comprises AAV2.

In some aspects, the AAV vector includes a splice acceptor site. In some aspects, the AAV vector includes a promoter. Any promoter known in the art can be used in the AAV vectors of this disclosure. In some embodiments, the promoter is an RNA Pol III promoter. In some aspects, the RNA Pol III promoter is selected from the group consisting of U6 promoter, H1 promoter, 7SK promoter, 5S promoter, adenovirus 2 (Ad2) VAI promoter, and any combination thereof. In some aspects, the promoter is the cytomegalovirus immediate early gene (CMV) promoter, EF1a promoter, Sv40 promoter, PGK1 promoter, Ubc promoter, human beta actin promoter, CAG promoter, TRE promoter, UAS promoter, Ac5 promoter, poly Hedrin promoter, CaMKIIa promoter, GAL1 promoter, GAL10 promoter, TEF promoter, GDS promoter, ADH1 promoter, CaMV35S promoter, or Ubi promoter. In certain aspects, the promoter comprises the U6 promoter.

In some aspects, the AAV vector contains a constitutively active promoter (constitutive promoter). In some aspects, the constitutive promoter is hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, cytomegalovirus (CMV), simian virus (e.g., SV40), papillomavirus, adenovirus Virus, human immunodeficiency virus (HIV), Rous sarcoma virus, retroviral long terminal repeat (LTR), murine stem cell virus (MSCV), and thymidine kinase promoter of herpes simplex virus.

In some aspects, the promoter is an inducible promoter. In some aspects, the inducible promoter is a tissue-specific promoter. In certain aspects, the tissue-specific promoter directs transcription of the coding region of the AAV vector in neurons, glial cells, or both neurons and glial cells.

In some aspects, the AAV vector comprises one or more enhancers. In some aspects, one or more enhancers are present in AAV alone or together with the promoters disclosed herein. In some aspects, the AAV vector includes a 3'UTR poly(A) tail sequence. In some aspects, the 3'UTR poly(A) tail sequence is selected from the group consisting of bGH poly(A), actin poly(A), hemoglobin poly(A), and any combination thereof. In some aspects, the 3'UTR poly(A) tail sequence comprises bGH poly(A).

In some aspects, the miR-485 inhibitors disclosed herein are administered with a delivery agent. Non-limiting examples of delivery agents that can be used include lipidoids, liposomes, lipoplexes, lipid nanoparticles, polymeric compounds, peptides, proteins, cells, nanoparticle mimetics, nanotubes, micelles, or conjugates. be done.

Accordingly, in some aspects, the disclosure also provides compositions comprising a miRNA inhibitor of the disclosure (ie, miR-485 inhibitor) and a delivery agent. In some embodiments, the delivery agent has the formula:
[WP]-L1-[CC]-L2-[AM] (Formula I)
or [WP]-L1-[AM]-L2-[CC] (Formula II)
(In the formula,
WP is the water-soluble biopolymer moiety;
CC is a positively charged carrier moiety,
AM is the adjuvant moiety;
L1 and L2 are independently optional linkers, comprising a cationic carrier unit with
Cationic carrier units form micelles when mixed with nucleic acids in an ionic ratio of about 1:1.

In some aspects, compositions comprising miRNA inhibitors (ie, miR-485 inhibitors) of the present disclosure interact with cationic carrier units via ionic bonds.

（式中、ｎは、１～１０００である）を有する。 In some aspects, the water-soluble polymer is poly(alkylene glycol), poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate) , poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazoline (“POZ”), poly(N-acryloylmorpholine), or any combination thereof . In some aspects, the water-soluble polymer comprises polyethylene glycol (“PEG”), polyglycerol, or poly(propylene glycol) (“PPG”). In some aspects, the water-soluble polymer has the formula:

(where n is 1-1000).

In some aspects, n is at least about 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about about 120, at least about 121, at least about 122, at least about 123, at least about 124, at least about 125, at least about 126, at least about 127, at least about 128, at least about 129, at least about 130, at least about 131, at least about 132 , at least about 133, at least about 134, at least about 135, at least about 136, at least about 137, at least about 138, at least about 139, at least about 140, or at least about 141. In some aspects, n is from about 80 to about 90, from about 90 to about 100, from about 100 to about 110, from about 110 to about 120, from about 120 to about 130, from about 140 to about 150, or from about 150 to about 160.

In some aspects, the water-soluble polymer is linear, branched, or dendritic. In some aspects, the cationic carrier moiety comprises one or more basic amino acids. In some aspects, the cationic carrier moieties are at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30 , at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least It contains 47, at least 48, at least 49, or at least 50 basic amino acids. In some aspects, the cationic carrier moiety comprises from about 30 to about 50 basic amino acids. In some aspects, basic amino acids include arginine, lysine, histidine, or any combination thereof. In some aspects, the cationic carrier moiety comprises about 40 lysine monomers.

（式中、Ｇ１及びＧ２のそれぞれは、Ｈ、芳香環、もしくは１～１０アルキルであるか、またはＧ１とＧ２はともに芳香環を形成し、ｎは１～１０である）を有する。 In some aspects, an adjuvant moiety can modulate an immune response, an inflammatory response, and/or a tissue microenvironment. In some aspects, the adjuvant moiety comprises imidazole derivatives, amino acids, vitamins, or any combination thereof. In some embodiments, the adjuvant moiety has the formula:

(wherein each of G1 and G2 is H, an aromatic ring, or 1-10 alkyl, or G1 and G2 together form an aromatic ring and n is 1-10).

In some aspects, the adjuvant moiety comprises a nitroimidazole. In some embodiments, the adjuvant moiety comprises metronidazole, tinidazole, nimorazole, dimetridazole, pretomanide, ornidazole, megazole, azanidazole, benznidazole, or any combination thereof. In some aspects, the adjuvant moiety comprises an amino acid.

（式中、Ａｒは、

であり、
Ｚ１及びＺ２のそれぞれは、ＨまたはＯＨである）を有する。 In some embodiments, the adjuvant moiety has the formula:

(wherein Ar is

and
each of Z1 and Z2 is H or OH.

（式中、Ｙ１及びＹ２のそれぞれは、Ｃ、Ｎ、Ｏ、またはＳであり、ｎは１または２である）を有する。 In some aspects, the adjuvant portion includes vitamins. In some aspects, the vitamin comprises a cyclic ring or cyclic heteroatom ring and a carboxyl or hydroxyl group. In some embodiments, the vitamin has the formula:

where each of Y1 and Y2 is C, N, O, or S and n is 1 or 2.

In some aspects, the vitamins are vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D2, vitamin D3, vitamin E, vitamin M, vitamin H , and any combination thereof. In some aspects, the vitamin is vitamin B3.

In some aspects, the adjuvant moieties are at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or containing at least about 20 vitamin B3. In some aspects, the adjuvant portion comprises about 10 vitamin B3.

In some aspects, the composition comprises a water-soluble biopolymer moiety having about 120 to about 130 PEG units, a cationic carrier moiety comprising polylysine having about 30 to about 40 lysines, and about and an adjuvant moiety having 5 to about 10 vitamin B3.

In some aspects, the composition comprises (i) a water-soluble biopolymer moiety having from about 100 to about 200 PEG units and (ii) from about 30 to about 40 lysines having amine groups, such as , about 32 lysines), (iii) about 15-20 lysines each having a thiol group (eg, about 16 lysines each having a thiol group), and (iv) vitamin B3. and about 30-40 lysines fused together (eg, about 32 lysines each fused to vitamin B3). In some aspects, the composition further comprises a targeting moiety, eg, a LAT1 targeting ligand, eg, phenylalanine, attached to the water-soluble polymer. In some aspects, thiol groups in the composition form disulfide bonds.

In some aspects, the composition comprises (1) (i) from about 100 to about 200 PEG units and (ii) from about 30 to about 40 lysines having amine groups (e.g., about 32 (iii) about 15-20 lysines each having a thiol group (eg, about 16 lysines each having a thiol group); and (iv) about 30 lysines fused to vitamin B3. and (e.g., about 32 lysines each fused to vitamin B3), and (2) a miR485 inhibitor (e.g., SEQ ID NO: 30) to inhibit miR485 The agent is entrapped within the micelle. In some aspects, the composition further comprises a targeting moiety, eg, a LAT1 targeting ligand, eg, phenylalanine, attached to the PEG unit. In some aspects, the thiol groups in the micelles form disulfide bonds.

The disclosure also provides micelles comprising a miRNA inhibitor of the disclosure (ie, miR-485 inhibitor, eg, SEQ ID NO:30), wherein the miRNA inhibitor and delivery agent are bound to each other.

In some aspects, the binding is covalent, non-covalent, or ionic. In some aspects, the positive charge of the cationic carrier portion of the cationic carrier unit is sufficient to form micelles when mixed with the miR-485 inhibitors disclosed herein in solution. , the overall ionic ratio of the positive charge of the cationic carrier portion of the cationic carrier unit in solution to the negative charge of the miR-485 inhibitor (or vector containing the inhibitor) is approximately 1:1.

In some aspects, the cationic carrier unit can protect the miRNA inhibitors of the present disclosure (i.e., miR-485 inhibitors) from enzymatic degradation (herein incorporated by reference in its entirety, 2020 See PCT Publication No. WO 2020/261227, published Dec. 30, 2020).

V. Pharmaceutical Compositions In some aspects, the present disclosure includes a miR-485 inhibitor disclosed herein (e.g., a polynucleotide or vector comprising a miR-485 inhibitor) suitable for administration to a subject Pharmaceutical compositions are also provided. Pharmaceutical compositions generally comprise a miR-485 inhibitor (eg, polynucleotide or vector) described herein and a pharmaceutically acceptable excipient or carrier for administration to a subject. Contain in suitable form. Pharmaceutically acceptable excipients or carriers are determined in part by the particular composition being administered and the particular method used to administer the composition.

Accordingly, a wide variety of suitable formulations of pharmaceutical compositions comprising miR-485 inhibitors of the present disclosure exist (see, eg, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18th ed. (1990)). reference). Pharmaceutical compositions are generally formulated as sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration.

VI. Kits The present disclosure provides miRNA inhibitors of the disclosure (e.g., polynucleotides, vectors, or pharmaceutical compositions disclosed herein) and, optionally, instructions for use, e.g., methods disclosed herein. Also provided are kits or articles of manufacture that include instructions for use according to. In some aspects, a kit or article of manufacture comprises a miR-485 inhibitor (eg, a vector, eg, an AAV vector, polynucleotide, or pharmaceutical composition of this disclosure) in one or more containers. In some aspects, a kit or article of manufacture comprises a miR-485 inhibitor (eg, a vector, eg, an AAV vector, polynucleotide, or pharmaceutical composition of the present disclosure) and a brochure. The miR-485 inhibitors disclosed herein (e.g., vectors, polynucleotides, and pharmaceutical compositions of the disclosure, or combinations thereof) can be packaged in one of the established kit formats well known in the art. Those skilled in the art will readily recognize that it can be easily incorporated.

The following examples are presented by way of illustration and not by way of limitation.

Example 1 Materials and Methods Unless otherwise specified, the examples described below employ one or more of the following materials and methods.

Human Tissue Centrocentral gyrus samples from patients with Alzheimer's disease (AD) and controls were purchased from Brainbank, The Netherlands. Information about these patients and controls is shown in Table 1.

Mice B6SJLF1/J (JAX#100012) and transgenic mice with five familial AD mutations (5XFAD) (#MMRRC#034848) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). 5XFAD mice carry mutant human amyloid precursor protein (APP) with Swedish (K670N, M671L), Florida (I716V), and London (V717I) mutations and two FAD mutations (M146L and L286V). Overexpressed with mutant human presenilin 1 (PS1). These transgenes are regulated by the neuronal Thy1 promoter. The genotype of 5XFAD mice was confirmed by PCR of tail DNA according to standard PCR conditions provided by The Jackson Laboratory. Four to five genotype-mixed mice per cage were housed on a 12 h light/12 h dark cycle with food and water available ad libitum. All animal care procedures were performed in accordance with Konyang University guidelines for the care and use of laboratory animals. Animal experiments were approved by the Konyang University Committee (permit number: P-18-18-A-01).

6-Hydroxydopamine (6-OHDA) mice (C57BL/6, 8 weeks old, 20-23 g) were obtained from KOATECH (Pyeontaek, Korea). These mice were housed in a controlled environment with free access to food and water.

Next Generation Sequencing Using Mouse Frontal Cortex NGS was performed on NovaSeq 6000 system (Illumina) by Theragen Etex Bio Institute (Seoul, Republic of Korea, worldwideweb.theragenetex.com/kr/bio). Libraries were constructed using the TruSeq Stranded mRNA Library Kit (Illumina). Data were then processed using 'unprocessed reads' in mRNA sequencing. Raw reads were aligned against GRCm38.96 (NCBI) using STAR aligner v2.7.1 for calculation of "RSEM" expression values (Dobin et al., Bioinformatics 29(1):15-21 (2013)). We implemented STAR aligner as the default option. The total number of reads for each sample was different and normalized by the TMM method. Thirteen mouse samples were treated similarly. All data are available at GEO (Gene Expression Omnibus, worldwideweb.ncbi.nlm.nih.gov/geo/) as GSE142633.

Using Public Databases, Reanalysis, and Network Analysis We used results by Weinberg et al. to identify miRNAs highly associated with cognitive impairment (Weinberg et al., Front Neurosci9:430 (2015) ). The 100 genes shown in Figure EV1 are extracted from Table 1 of Weinberg et al. We took the log2 of Weinberg et al.'s results, ordered them, and marked the target miRNAs. The 'miRDB' was used to search for specific genes targeting miRNAs (Wong et al., Nucleic Acids Res 43:D146-52 (2015)). The "Genecard" database was used to search for genes associated with diseases or biological conditions (Rebhan et al., Bioinformatics 14(8):656-64 (1998)). The results in Figure EV2A show search results in August 2019 using the keywords “Inflammation”, “Amyloid beta degradation” and “Alzheimer”. We used the R 'VennDiagram' package for Venn diagram analysis. The 'GeneMAINA' (version 3.5.1) package of Cytoscape (version 3.7.1) was used for analysis of protein-protein interactions (Franz et al., Nucleic Acids Res 46(W1): W60-W64 (2018)). We used 265 common genes, including hsa-miR-485-3p target genes and Alzheimer's-associated genes, as input for protein interaction analysis. Of these, 139 genes interacted without flanking genes. In addition, 9 genes are highly associated with cranial nervous system diseases (including AD) while low expression has been reported in patient groups or mouse models of dementia.

Intracerebroventricular Injection of miR-485 Inhibitors miR485-3p Antisense Oligonucleotide (ASO) (i.e. miR-485 Inhibitor) (AGAGAGGAGAGCCGUGUAUGAC) (SEQ ID NO: 30) and Control Oligonucleotide (“miR-control”) (CCTTCCCTGAAGGTTCCTCCTT) (SEQ ID NO: 61) was synthesized by Integrated DNA Technologies (USA). All animals were initially anesthetized with 3-5% isoflurane in oxygen and then fixed on a brain stereotaxic frame (JeongDo). For intracerebroventricular (ICV) injections, miR-485 inhibitors or non-targeting control oligonucleotides were formulated in vivo with jetPEI reagent (Polyplus). miR-485 inhibitors (1.5 μg) or control oligonucleotides formulated with jetPEI reagent in vivo were injected at 1.5 μL/min via a 10 μL Hamilton syringe (26 gauge blunt needle). A volume of 5 μL of miR-485 inhibitor and control oligonucleotides were injected intracerebroventricularly (ICV) into 10-month-old 5XFAD mice. A miR-485 inhibitor or a non-targeting control oligonucleotide was administered weekly for two weeks. The intracerebroventricular (ICV) location was identified using the coordinates from Bregma below. AP = -0.2 mm, L = ±1.0 mm, ventral (V) = -2.5 mm

Intravenous injection of a miR-485 inhibitor miR485-3p antisense oligonucleotide (ASO) (i.e. miR-485 inhibitor) (AGAGAGGAGAGCCGUGUAUGAC) (SEQ ID NO: 30) or control oligonucleotide (“miR control”) (CCTTCCCTGAAGGTTCCTCCTT) ( SEQ ID NO: 61) was loaded into nanoparticles with a PEGylated shell, a crosslinked core and one or more brain targets. In some embodiments, ASOs are fluorescently labeled (eg, Cy5.5) to allow tracking using in vivo imaging. Fluorescent images were taken as pre-injection images prior to injection of micelles or ASO. ASO-loaded nanoparticles (25 μg ASO) were administered intravenously (tail vein injection) to mice and fluorescent images of the mice were taken at desired times using the IVIS in vivo imaging system. Unless otherwise indicated, mice received a single dose of ASO-loaded nanoparticles. Fluorescence images were observed for up to 16 hours and the time-dependent fluorescence intensity of ASO-loaded micelles was compared to mice injected with naked ASO. ASO-loaded micelles and fluorescence images of ASO were taken as the distribution of ASO to compare the biodistribution behavior of the two groups.

All animals were initially anesthetized with 3-5% isoflurane in oxygen and then fixed on a brain stereotaxic frame (JeongDo). For intracerebroventricular (ICV) injections, miR-485 inhibitors or non-targeting control oligonucleotides were formulated in vivo with jetPEI reagent (Polyplus). miR-485 inhibitors (1.5 μg) or control oligonucleotides formulated with jetPEI reagent in vivo were injected at 1.5 μL/min via a 10 μL Hamilton syringe (26 gauge blunt needle). A volume of 5 μL of miR-485 inhibitor and control oligonucleotides were injected intracerebroventricularly (ICV) into 10-month-old 5XFAD mice. A miR-485 inhibitor or a non-targeting control oligonucleotide was administered weekly for two weeks. The intracerebroventricular (ICV) location was identified using the coordinates from the previous section below. AP=−0.2 mm, L=±1.0 mm, ventral (V)=−2.5 mm.

Glial and Cortical Neuronal Cell Cultures and Transfection Mouse primary mixed glial cells were cultured from the brain cortex of 1-3 day old C57BL/6 mice. Brain cortices were excised and broken into single cell suspensions by pipetting. Single cell suspensions were then seeded into 6-well plates precoated with 0.05 mg/ml poly-D-lysine (PDL), 25 mM glucose, 10% (vol/vol) heat-inactivated fetal bovine serum, Cultured for 2 weeks in DMEM medium supplemented with 2 mM glutamine and 1,000 units/mL penicillin-streptomycin (P/S). Primary cortical neurons were cultured from day 17 mouse embryos. Briefly, cortices were excised, incubated in ice-cold HBSS (Welgene, LB003-02) solution, and dissociated in accumax (Sigma, Catalog No. A7089) for 15 minutes at 37°C. Cultures were washed twice in HBSS. Mouse neurons were resuspended in neurobasal medium (Gibco, Cat. No. 21103049) containing 2% B27 (Gibco, Cat. No. 17504), 1% sodium pyruvate, and 1% P/S. Cells were filtered through a 70 μM cell strainer (SPL, 93070) and maintained at 37° C. in a humidified 5% CO 2 incubator. After changing the medium every 3 days and then culturing in vitro for 12-13 days, the cells were used for experiments. Primary glial cells or cortical neurons were injected with 100 nM miR control, 100 nM has-miR485-3p mimetic, or 100 nM miR-485 inhibitor using TRANSIT-X2® transfection reagent (Mirus Bio). was transfected.

Luciferase Assay The 3′UTR of human SIRT1 containing the target site of miR-485-3p was amplified from cDNA by PCR amplification and inserted into the psiCHECK2 vector (Promega, Cat#C8021). HEK293T cells in 96-well plates were transfected with psiCHECK2-Sirt1-3′UTR wild type (WT) or psiCHECK2-Sirt1-3′UTR mutant (MT) and miR-485-3p with Lipofectamine 2000 (Invitrogen, Cat. 027) were used to co-transfect. Cells were harvested after 48 hours and luciferase reporter activity was measured using the Dual Luciferase Assay System (Promega, Catalog No. E1910). Three independent experiments were performed in triplicate.

The 3'UTR of human CD36 containing the target site of miR-485-3p was amplified from cDNA by PCR amplification and inserted into the pMir-Target vector (Addgene). HEK293T cells in 96-well plates were incubated with pMir-CD36-3'UTR WT or pMir-CD36-3'UTR MT with pRL-SV40 vector (Addgene) and miR-485-3p with Lipofectamine 2000 (Invitrogen, Cat. No. 11668-027) were used to co-transfect. Cells were harvested 24-48 hours later and luciferase reporter activity was measured using the Dual Luciferase Assay System (Promega, Catalog No. E1910). Three independent experiments were performed in triplicate.

In Vitro Binding Assays Streptavidin magnetic beads (Invitrogen, Catalog No. 11205D) were prepared for in vitro binding assays as follows. Beads (50 μL) were washed 5 times with 500 μL of 1×B&W buffer (5 mM Tris-HCl, pH 7.4; 0.5 mM EDTA; 1 M NaCl). After removing the supernatant, the beads were incubated with 500 μL of 1×B&W buffer containing 100 μg of yeast tRNA (Invitrogen, Cat#AM7119) for 2 hours at 4°C. The beads were washed twice with 500 μL of 1×B&W buffer and incubated with 200 μL of 1×B&W buffer containing 400 pmol of biotin-miR-485-3p for 10 minutes at room temperature. The supernatant was removed and the beads were washed twice with 500 μL of 1×B&W buffer and collected on a magnetic stand. The miRNA-coated beads were incubated with 500 μL of 1×B&W buffer containing 1 μg of in vitro transcribed target mRNA at 4° C. overnight. The next day, the beads were washed five times with 1 ml of 1×B&W buffer and then resuspended in 200 μL of RNase-free water. Bound RNA was extracted using the QiaZol Lysis reagent (Qiagen, catalog number 79306) according to the manufacturer's instructions. Extracted RNA was quantified by the StepOnePlus real-time PCR system (Applied Biosystems, REF: 4376592).

Western Blot Brain tissue, primary glial cells or cortical neurons were homogenized for 30 minutes on ice in ice-cold RIPA buffer (iNtRON Biotechnology) containing a protease/phosphatase inhibitor cocktail (Cell Signaling Technology, cat#5872). . The lysate was centrifuged at 13000 rpm for 15 minutes at 4° C. and the supernatant collected. Samples were separated by SDS polyacrylamide gel electrophoresis, transferred to PVDF membranes, and tested with the following primary antibodies: rabbit anti-PGC-1α (Abcam, catalog number ab54481, 1:1000), rabbit anti-APP (Cell Signaling Technology, 2452, 1:1000), mouse anti-sAPPα (IBL, Cat.No. 11088, 1:1000), mouse anti-sAPPα (IBL, cat.No. 10321, 1:1000), rabbit anti-Adam10 (Abcam, cat.No.ab1997, 1 :100), mouse anti-CTFs (Biolegend, Cat. No. SIG-39152, 1:1000), rabbit anti-β-amyloid (1-42) (Cell Signaling Technology, Cat. No. 14974, 1:1000), rabbit anti-BACE1 (Abcam , cat#ab2077, 1:1000), mouse anti-NeuN (Millipore, #MAB377, 1:1000), rabbit anti-cleaved caspase-3 (Cell Signaling Technology, cat#9664, 1:1000), mouse anti-GFAP (Merck, Cat# MAB360, 1:1000), rabbit anti-IL-1β (abcam, Cat#9722, 1:1000), rabbit anti-NF-kB(p65) (Cell Signaling Technology, Cat#8242, 1:1000), goat anti Iba1 (Abcam, cat#ab5076, 1:1000), rabbit anti-SIRT1 (Abcam, cat#04-1557), mouse anti-TNF-alpha (Santa Cruz, cat#sc-52746), antibody actin (Santa Cruz, cat# sc-47778). Results were visualized using an enhanced chemiluminescence system and quantified by densitometry analysis (Image J software, NIH). All experiments were performed independently at least three times.

To measure tyrosine hydroxylase expression in brain tissue (see Example 15), brain tissue was treated with ice-cold RIPA buffer (iNtRON) containing a protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Catalog No. 5872). Biotechnology) on ice for 30 minutes. The lysate was centrifuged at 13000 rpm for 15 minutes at 4° C. and the supernatant collected. Samples were separated by SDS polyacrylamide gel electrophoresis, transferred to PVDF membranes, and treated with the following primary antibodies: rabbit anti-tyrosine hydroxylase (TH; 1:2000; Pel-Freez, Brown Beer, Wisconsin, USA), and Incubated with mouse anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, Calif., USA). Membranes were then incubated with secondary antibodies for 1 hour at room temperature and bands were finally detected using Western blot reagents (Thermo Fisher Scientific, Rockford, Ill., USA). For quantitative analysis, the density of each band was measured using a Computer Imaging Device and accompanying software (Fuji Film, Tokyo, Japan), and the levels were normalized to the housekeeping protein band density for each sample. expressed quantitatively as All experiments were performed independently at least three times.

Extraction of soluble and insoluble Aβ Brain tissue samples were homogenized on ice with RIPA buffer containing protease/phosphatase inhibitors and then centrifuged at 12000 rpm for 15 minutes. The supernatant was collected. To obtain the insoluble fraction from brain tissue, the brain lysate pellet was thawed in insoluble extraction buffer [50 mM Tris-HCl (pH 7.5) + 2% SDS] containing protease/phosphatase inhibitor cocktail for 30 min on ice. . The lysate was centrifuged at 13000 rpm for 15 minutes at 4°C. Proteins were quantified using the Bicinchoninic Acid (BCA) Assay Kit (Bio-Rad Laboratories, Catalog No. 5000116) and adjusted to the same final concentrations. After denaturation, lysates were processed for Western blotting to measure insoluble Aβ.

Immunohistochemistry For immunohistochemistry, brains of 5XFAD injected with miR-485 inhibitors or control oligonucleotides were excised, post-fixed and embedded in paraffin. Coronal sections (10 μm thick) were cut through the infarct using a microtome and mounted on glass slides. Paraffin was removed and sections were washed with PBS-T and blocked with 10% bovine serum albumin for 2 hours. The following primary antibodies were then added: purified mouse anti-beta amyloid, 1-16 (Biolegend, #803001, 1 μg/ml), rabbit anti-beta amyloid (1-42) (Cell Signaling Technology, #14974s, 1:100). ), rabbit anti-Iba-1 (Wako, #019-19741, 2 μg/ml), goat anti-Iba-1 (Abcam, #ab5076, 2 μg/ml), rabbit anti-CD68 (Abcam, #ab125212, 1 μg/ml), Rabbit anti-GFAP (Abcam, #ab16997, 1:100), mouse anti-GFAP (Millipore, #MAB360, 1:500), rat anti-CD36 (Abcam, #ab80080, 1:100), mouse anti-TNF-alpha (Santa Cruz , #sc-52746, 1:100), rabbit anti-IL-1β (Abcam, #ab9722, 1 μg/ml), rabbit anti-cleaved caspase-3 (Cell Signaling Technology, #9662S, 1:300), mouse anti-NeuN (Millipore, #MAB377, 10 μg/ml). Images were acquired using a confocal microscope (Leica DMi8). Relative band intensities were normalized to actin using ImageJ software (NIH).

Thioflavin S Staining For Thioflavin S (ThS) staining, sliced brains were stained with filtered 1% Thioflavin S aqueous solution for 8 minutes. Sections were then rinsed with 80%, 95% ethanol and washed three times with distilled water. Brain slices were then mounted and slides were dried overnight in the dark. Images were taken with a Leica fluorescence microscope.

Preparation of Aβ ^1-42 Fibrils Aβ _1-42 hexafluoroisopropanal (HFIP) peptide (#AS-64129) was obtained from AnaSpec (Fremont, Ca, USA). Aβ1-42 fibrils were prepared as described above (Coraci et al., American J of Pathology 160(1):101-12 (2002).). To form fAβ synthetic human Aβ _1-42 , Aβ _1-42 , HFIP peptide was dissolved in DMSO to a stock concentration of 5 mM. Stock solutions were then diluted to 100 μM in serum-free DMEM and incubated at 37° C. for 72 hours. Filamentous Aβ (fAβ) was confirmed by SDS-PAGE.

In vitro phagocytosis assay (ELISA and immune cell staining)
BV2 microglial cells (2×10 ⁵ ) were seeded in 6-well plates overnight. Cells were transfected using TRANSIT-X2® Transfection Reagent (Mirus Bio, Catalog No. MIR6000) according to the manufacturer's instructions and treated with fAβ for 4 hours at a final concentration of 1 μM. Anti-CD36 antibodies were added to the culture medium along with fAβ when applicable. After 4 hours, medium was harvested from BV2 microglia. Levels of human Aβ(1-42) in supernatants were measured using the Human Aβ42 ELISA Kit (Invitrogen, Catalog No. KHB3441) according to the manufacturer's instructions.

Furthermore, phagocytosis of glial cells was confirmed by fluorescence microscopy. After coating the coverslips with poly-l-lysine, 8×10 ⁴ human primary glial cells were seeded per coverslip and placed overnight in wells of a 24-well plate. Primary glial cells were transfected with TRANSIT-X2® transfection reagent (Mirus Bio) according to the manufacturer's instructions and incubated in unlabeled fAβ for 4 hours at a final concentration of 1 μM. After 4 hours of incubation, cells were washed with cold PBS. To measure Aβ uptake, primary glial cells were then fixed with 100% methanol for 1 hour at −20° C., washed with PBS-T, and treated with mouse anti-β-amyloid 1-16, rabbit anti-GFAP (abcam, #ab16997). , 1:100), and rabbit anti-Iba-1 antibody (Wako, #019-19741, 2 μg/ml) at 4°C.

FACS Analysis All staining steps were performed in the dark and blocked with BD Fc Block. Primary glial cells were incubated with the following antibodies: Alexa488-conjugated anti-mouse CD36 (Biolegend, Cat#102607, 5 μg/ml) or isotype control Ab (Biolegend, Cat#400923, 5 μg/ml) for 30 minutes at 4°C. dyed. After 30 minutes, cells were washed with FACS buffer (PBS+1%). Data were analyzed with CellQuest (BD Bioscience) and FlowJo software (Treestar) packages.

Real-time PCR
Total RNA was isolated using the Isolation of small and large RNA kit (Macherey Nagel, Dfiren). cDNA was synthesized using the miScript II RT Kit (Qiagen, Hilden, Germany). For analysis, expression of miR-485-3p was performed by TaqMan miRNA analysis using TOPREAL™ qPCR 2X PreMIX (Enzynomics, Korea) on CFX Connect system (Bio-Rad). Real-time PCR measurements of individual cDNAs were performed using SYBR green and TaqMan probes to measure double-stranded DNA formation with the Bio-Rad real-time PCR system. The following primers were used. Probe: FAM-CGAGGTCGACTTCCTAGA-NFQ. (SEQ ID NO:51) miR-485-3p forward: 5′-CATACACGGCTCTCCTCTCTAAA-3′ (SEQ ID NO:52); mouse primer: actin forward: 5′-TCCTGTGGCATCCATGAAAC-3′ (SEQ ID NO:53), reverse: 5′-CAATGCCTGGGTACATGGTG -3′ (SEQ ID NO: 54); TNF forward: 5′-CCAAGTGGAGGAGCAGCT-3′ (SEQ ID NO: 55), reverse: 5′-GACAAGGTACAACCCATCGG-3′ (SEQ ID NO: 56); IL-1β forward: 5′-TTCGACACATGGGATAACGAGG- 3′ (SEQ ID NO: 57), reverse: 5′-TTTTTGCTGTGAGTCCCGGAG-3′ (SEQ ID NO: 58); miR-16 forward: 5′-CAGCCTAGCAGCACGTAAAT-3′ (SEQ ID NO: 59); reverse: 5′-GAATCGAGCACCAGTTACG-3′ (SEQ ID NO: 60); levels of miR-16 were used for normalization. Relative gene expression was analyzed by the 2-ΔΔct method.

ELISA (TNF-α and IL-1β)
Primary mixed glial cells (2×10 ⁵ ) were seeded overnight in 6-well plates. Cells were treated with miR-485 inhibitors and mouse α-synuclein PFF (aggregated) at a final concentration of 1 μg/ml for 18 hours. After 18 hours, medium was harvested from primary mixed glial cells. The levels of TNF-a and IL-1b in the supernatant were measured by mouse TNF-a ELISA kit (R&D system, catalog number MTA00B) and mouse IL-1b ELISA kit (R&D system, catalog number MLB00C). ELISA was performed according to the manufacturer's instructions.

Behavioral tests (Y-maze and passive avoidance)
The Y-maze consisted of three black opaque plastic arms (30 cm x 8 cm x 15 cm) oriented 120° to each other. The 5XFAD mouse was centered and allowed to explore all three arms. The number of arm entries and trials (alternating behavior is entry into three separate arms 10 cm from the center) was recorded and the alternation behavior rate was calculated. Entry was defined as having all three limbs in the arms of the Y-maze. The alternation rate was defined as the number of trials divided by the number of arm entries minus two multiplied by 100.

The passive avoidance test chamber was divided into white (light) and black (dark) compartments (41 cm x 21 cm x 30 cm). A 60 W bulb was placed in the light compartment. The floor (of the darkroom) contained a number of (2 mm) stainless steel rods spaced at 5 mm intervals. The test was conducted in 3 days. On day 1, mice are habituated in the light compartment for 5 minutes. The second day will be a training period. The test consists of two steps. In the first step, each mouse is placed in the light compartment and moved twice to the dark compartment. One hour after the first step, each mouse is placed in the light compartment. Thirty seconds later, the door separating the two compartments was opened, and after the mice entered the dark compartment, the door was closed and an electric foot shock (0.3 mA/10 g) was delivered from the grid floor for 3 seconds. Avoidance was considered learned if the mouse did not enter the dark compartment for more than 5 minutes, and this training was performed up to 5 times. Twenty-four hours after the training trial, mice were placed in the light room for testing. Latency was defined as the time for mice to enter the darkroom after the door separating the two compartments was opened. The time for the mouse to enter the dark compartment and exit the light compartment was defined as TDC (time spent in dark compartment).

Behavioral test (rotarod)
Mice were trained on a rotarod apparatus (rod diameter 3 cm) at a fixed speed of 10 rpm for 600 seconds once a day for 3 days. Performance on the rod was evaluated at a constant acceleration rate of 4-40 rpm for 300 seconds. Two consecutive trials were performed with an interval of 60 minutes.

Behavioral test (hang wire test)
In the motor coordination wire hang test, mice were tested on a taut metal wire 2 mm thick and 55 cm long. A custom hanging device consisting of a 60 cm long black polystyrene box was constructed into which the mouse would fall. The latency for the mouse to hang and fall off the wire was recorded by measuring the longest hanging time in 3 trials for each mouse.

Behavioral test (pole test)
The pole test assesses an animal's alertness and can be used as a measure of bradykinesia. Mice were placed head-up on top of a textured pole (8 mm diameter, 55 cm height). The total time for each mouse to reach the floor from the vertex was measured as performance. Prior to the actual testing, mice were trained for 5 trials per day for 3 days and locomotor activity of each mouse was performed 5 times 6 days after intravenous injection of 6-OHDA and miR-485 inhibitors. was evaluated as the mean of the trials.

Behavioral test (balance beam test)
Mice were mounted on a balance beam apparatus 0.5 cm wide and 1 m long. The balance beam used consisted of a 50 cm high transparent Plexiglas structure with a dark rest box at the end of the track. Mice were trained on the beam three times in the morning and allowed at least 15 minutes of rest between trials. Mice were placed in the dark resting box for at least 10 seconds before being returned to their home cages. Mice were then retested at least 2 hours after the training session in the afternoon. Mouse performance was recorded during the test session. The test consisted of 3 trials with a rest period of at least 10 minutes between trials. The total number of foot slides was manually calculated for the last of the three trials. SOD1G93A mutant mice were tested 44 or 48 days after injection of PBS or miR-485 inhibitors.

Data Analysis All data are presented as mean±SD. NGS data were analyzed using R (version 3.5.2). Statistical significance of values obtained in two different groups was determined using the unpaired t-test. Statistical tests were performed using GraphPad Prism 5 or 8 (GraphPad Software, La Jolla, Calif.). Statistical significance between two groups was analyzed by two-way ANOVA and unpaired t-test. Behavioral tests were evaluated by nonparametric statistical methods.

Example 2: Preparation of miR-485 inhibitors (a) Synthesis of alkyne-modified tyrosines: Tissue-specific targeting moieties (TM, Figure 1) were prepared as intermediates for the synthesis of alkyne-modified tyrosines.

N-(tert-butoxycarbonyl)-L-tyrosine methyl ester (Boc-Tyr-OMe) (0.5 g, 1.69 mmol) and K ₂ CO ₃ (1.5 eq, 2 .54 mmol) was added dropwise to propargyl bromide (1.2 eq, 2.03 mmol). The reaction mixture was heated at 60° C. overnight. After reaction, the reaction mixture was extracted using water:ethyl acetate (EA). The organic layer was then washed using a brine solution. The crude was purified by flash column (10% EA in hexanes). The resulting product was then dissolved in 1,4-dioxane (1.0 ml) and 6.0 M HCl (1.0 ml). The reaction mixture was heated at 100° C. overnight. Dioxane was then removed and extracted with EA. Aqueous NaOH (0.5 M) solution was added to the mixture until the pH value was 7. The reactants were concentrated on an evaporator and centrifuged at 12,000 rpm at 0°C. The precipitate was washed with deionized water and lyophilized.

(b) Synthesis of poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL): This synthetic step yields a water-soluble biopolymer (WP) of cationic carrier units of the present disclosure and a cationic carrier (WP) CC) were prepared (see Figure 1).

Poly(ethylene glycol)-b-poly(L-lysine) was synthesized by ring-opening polymerization of Lys(TFA)-NCA using monomethoxy PEG (MeO-PEG) as macroinitiator. Briefly, MeO-PEG (600 mg, 0.12 mmol) and Lys(TFA)-NCA (2574 mg, 9.6 mmol) were dissolved separately in DMF and DMF (or NMP) containing 1M thiourea. The Lys(TFA)-NCA solution was added dropwise to the MeO-PEG solution via a microsyringe and the reaction mixture was stirred at 37° C. for 4 days. The reaction bottle was purged with argon and vacuum. All reactions were performed under an argon atmosphere. After reaction, the mixture was precipitated in excess diethyl ether. The precipitate was redissolved in methanol and reprecipitated in cold diethyl ether. The precipitate was then filtered and a white powder was obtained after drying under reduced pressure. To deprotect the TFA group of PEG-PLL(TFA), the following steps were performed.

MeO-PEG-PLL(TFA) (500 mg) was dissolved in methanol (60 mL) and 1N NaOH (6 mL) was added dropwise to the polymer solution under stirring. The mixture was kept at 37° C. for 1 day with stirring. The reaction mixture was dialyzed against 10 mM HEPES four times and distilled water. A white powder of PEG-PLL was obtained after lyophilization.

(b) Synthesis of azido-poly(ethylene glycol)-b-poly(L-lysine) (N ₃ -PEG-PLL): This synthetic step yields a water-soluble biopolymer (WP) of cationic carrier units of the present disclosure. and a cationic carrier (CC) were prepared (see Figure 1).

Azido-poly(ethylene glycol)-b-poly(L-lysine) was synthesized by ring-opening polymerization of Lys(TFA)-NCA with azide-PEG (N ₃ -PEG). Briefly, N ₃ -PEG (300 mg, 0.06 mmol) and Lys(TFA)-NCA (1287 mg, 4.8 mmol) were separately dissolved in DMF and DMF (or NMP) containing 1M thiourea. The Lys(TFA)-NCA solution was added dropwise to the N ₃ -PEG solution via a microsyringe and the reaction mixture was stirred at 37° C. for 4 days. The reaction bottle was purged with argon and vacuum. All reactions were performed under an argon atmosphere. After reaction, the mixture was precipitated in excess diethyl ether. The precipitate was redissolved in methanol and reprecipitated in cold diethyl ether. The precipitate was then filtered and a white powder was obtained after drying under reduced pressure. To deprotect the TFA group of PEG-PLL(TFA), the following steps were performed.

N ₃ -PEG-PLL (500 mg) was dissolved in methanol (60 mL) and 1N NaOH (6 mL) was added dropwise to the polymer solution with stirring. The mixture was kept at 37° C. for 1 day with stirring. The reaction mixture was dialyzed against 10 mM HEPES four times and distilled water. A white powder of N ₃ -PEG-PLL was obtained after lyophilization.

(c) Synthesis of (methoxy or) azido-poly(ethylene glycol)-b-poly(L-lysine/nicotinamide/mercaptopropanamide) (N ₃ -PEG-PLL(Nic/SH)): In this step, A tissue-specific adjuvant moiety (AM, see Figure 1) was attached to the WP-CC component of the cationic carrier unit of the present disclosure. The tissue-specific adjuvant moiety (AM) used for the cationic carrier unit was nicotinamide (vitamin B3). This step yields the WP-CC-AM component of the cationic carrier unit shown in FIG.

Azido-poly(ethylene glycol)-b-poly(L-lysine/nicotinamide/mercaptopropanamide) (N ₃ -PEG-PLL(Nic/SH)) was reacted with N ₃ -PEG- Synthesized by chemical modification of PLL and nicotinic acid. N ₃ -PEG-PLL (372 mg, 25.8 μmol) and nicotinic acid (556.7 mg, 1.02 equivalents to NH2 of PEG-PLL) were separately added to a mixture of deionized water and methanol (1:1). dissolved in EDC.HCl (556.7 mg, 1.5 equivalents relative to _NH2 of _N3- PEG-PLL) was added to the nicotinic acid solution and NHS (334.2 mg, 1.5 equivalents relative to NH2 of PEG-PLL). ) was added stepwise to the mixture.

The reaction mixture was added to the N ₃ -PEG-PLL solution. The reaction mixture was maintained at 37° C. for 16 hours with stirring. After 16 hours, 3,3′-dithiodipropionic acid (36.8 mg, 0.1 eq.) was dissolved in methanol, EDC.HCl (40.3 mg, 0.15 eq.), and NHS (24.2 mg, 0.15 eq.). 0.15 equivalents) were each dissolved in deionized water. NHS and EDC.HCl were then sequentially added to the 3,3'-dithiodipropionic acid solution. After the crude N ₃ -PEG-PLL(Nic) solution was added, the mixture was stirred at 37° C. for 4 hours.

For purification, the mixture was dialyzed against methanol for 2 hours and DL-dithiothreitol (DTT, 40.6 mg, 0.15 equiv) was added followed by 30 minutes activation.

To remove DTT, the mixture was dialyzed sequentially against methanol, 50% methanol in deionized water, deionized water.

d) Synthesis of phenylalanine-poly(ethylene glycol)-b-poly(L-lysine/nicotinamide/mercaptopropanamide) (Phe-PEG-PLL(Nic/SH)): In this step, a tissue-specific targeting moiety (TM) was attached to the WP-CC-AM component synthesized in the step above. The TM component (phenylalanine) was prepared by reaction of the intermediate prepared in step (a) with the product of step (c).

To target brain endothelial tissue in blood vessels, phenylalanine was used as the LAT1-targeting amino acid, and the reaction between N ₃ -PEG-PLL (Nic/SH) and alkyne-modified tyrosine in the presence of copper catalysts. was introduced by the click reaction of Briefly, N ₃ -PEG-PLL(Nic/SH) (130 mg, 6.5 μmol) and alkyne-modified phenylalanine (5.7 mg, 4.0 equiv) were combined in deionized water (or 50 mM sodium phosphate buffer). ). CuSO4·H2O (0.4 mg, 25 mol%) and Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, 3.4 mg, 1.2 equiv.) were then dissolved in deionized water and N _-PEG was dissolved. -PLL (Nic/SH) solution was added. Sodium ascorbate (3.2 mg, 2.5 eq) was then added to the mixture. The reaction mixture was kept stirring at room temperature for 16 hours. After reaction, the mixture was transferred to a dialysis membrane (MWCO=7,000) and dialyzed against deionized water for 1 day. The final product was obtained after lyophilization.

(E) Polyion Complex (PIC) Micelle Formulations - After preparing the cationic carrier units of the present disclosure as described above, micelles were made. The micelles described in this example consist of cationic carrier units combined with an antisense oligonucleotide payload.

Nano-sized PIC micelles were prepared by mixing MeO-PEG-PLL(Nic) or Phe-PEG-PLL(Nic) with miRNA. PEG-PLL (Nic) was dissolved in HEPES buffer (10 mM) at a concentration of 0.5 mg/mL. A solution of miRNA (22.5 μM) in RNAse-free water was then added to a miRNA inhibitor (SEQ ID NO: 2-30) (eg, AGAGAGGAGAGCGUGUAUUGAC; SEQ ID NO: 30) at a 2:1 (v/v) ratio of polymer to polymer. was mixed with the polymer solution so that

The mixing ratio of polymer and anti-miRNA was determined by optimizing the micelle formation conditions, ie the ratio of amine in polymer (carrier of the present disclosure) and phosphate in anti-miRNA (payload). The mixture of polymer (carrier) and anti-miRNA (payload) was mixed vigorously by multi-vortex at 3000 rpm for 90 seconds and kept at room temperature for 30 minutes to stabilize the micelles.

Micelles (10 μM anti-miRNA concentration) were stored at 4° C. prior to use. MeO-micelles or Phe-micelles were prepared using the same method, and micelles containing different amounts of Phe (25%-75%) were also prepared by mixing both polymers during micelle preparation.

Example 3 Analysis of SIRT1 Expression in Alzheimer's Disease A previous study reported that levels of SIRT1 are decreased in the brains of human AD patients and that this decrease affects the progression of early to late AD. (Julien et al, 2009, Lutz et al, 2014). Initiating the evaluation of the potential therapeutic effects of the miR-485 inhibitors disclosed herein, SIRT1 expression was assessed in postmortem brain (precentral gyrus) samples from Alzheimer's disease (AD) patients. As shown in Figures 2A and 2B, SIRT1 protein levels were significantly reduced in AD patient brains compared to normal human brains.

To confirm the above results, SIRT1 expression was evaluated in an established AD animal model, ie, transgenic mice with five familial AD mutations (5XFAD). As shown in FIG. 1C, there was no significant difference in SIRT1 expression in 6-month-old AD mice compared to wild-type control animals. However, there was a significant reduction in SIRT1 expression in 11 month old AD mice (see Figure 2C). SIRT1 expression was progressively decreased in 5XFAD aged mice (Fig. 2D).

The above results corroborate previous studies, suggesting that SIRT1 expression is downregulated in AD and that SIRT1 may play a role in the pathogenesis of AD.

Example 4 Analysis of Efficacy of miR-485 Inhibitors in Regulating Expression of miR485-3p Further analysis was performed for overexpressed miRNAs. As shown in FIG. 3A, miR485-3p expression was significantly higher in precentral precentral tissue of AD patients compared to normal healthy tissue. No significant difference was observed for other SIRT1-related miRNAs, including miR485-5p (see Figure 3B).

Using published algorithms (eg, Targetscan and miRbase programs), miR-485-3p was then predicted to have a binding site within the 3'UTR of SIRT1. To confirm this, the ability of human miR485-3p mimics and inhibitors to inhibit miR485-3p expression in mice was assessed. As shown in FIG. 4, using real-time PCR analysis, a significant reduction in miR485-3p expression was observed when mouse primary cortical neurons were transfected with a human miR485-3p inhibitor.

This result indicates that miR485 inhibitors can reduce and/or inhibit the expression of miR485-3p.

Example 5 Analysis of Regulation of SIRT1 by miR-485 Inhibitors a) human miR485-3p, or (iii) one of the miR485-3p inhibitors. SIRT1 expression in transfected cells was then assessed. As shown in FIGS. 5A and 5B, SIRT1 protein expression was reduced in miR485-3p-transfected primary cortical neurons compared to miR-control-transfected neurons. In contrast, primary cortical neurons transfected with the miR inhibitors disclosed herein expressed significantly higher levels of SIRT1 protein. SIRT1 expression also appeared to correlate with PGC-1α expression, as shown in FIGS. 5A and 5B.

These results demonstrate that miR485 inhibitors of the present disclosure can increase SIRT1 expression by modulating miR485-3p expression. These results further demonstrate that the miR inhibitors disclosed herein can also be useful in increasing the expression of PGC-1α protein in cells.

Example 6: Analysis of binding of miR485-3p to SIRT1 To confirm the target site for miR485-3p on SIRT1, luciferase of the 3'UTR of SIRT1 containing wild-type or mutant sequences of potential miR485-3p sites. A reporter plasmid was constructed (see Figure 6A). HEK293T cells were then transfected with these plasmids and promoter activity was measured in transfected cells. As shown in FIG. 6B, in miR485-3p-transfected cells, wild-type promoter activity was significantly reduced, but the mutant did not differ.

Next, we assessed the physical binding of miR485-3p to the 3'UTR of SIRT1 using an in vitro binding assay. Briefly, streptavidin-miR483-3p-coated magnetic beads were incubated with in vitro transcribed wild-type and mutant 3′UTR of SIRT1, respectively. Bound RNA was eluted and quantified by real-time PCR. Total binding was calculated using the following formula: relative binding=100×2 ^{((adjusted input)−(Ct IP))} /100×2 ^{((adjusted input)−(Ct WT))} . The relative binding efficiency was significantly lower for the mutant seed sequence containing the 3'UTR compared to the wild-type seed sequence (the site to which the miRNA binds) (see Figure 6C).

Taken together, the above results indicate that miR485-3p can directly target the 3'UTR of SIRT1 and that this interaction can negatively regulate SIRT1 expression.

Example 7 Analysis of miR-485 Inhibitors in Beta-Amyloid (Aβ) Plaque Formation To investigate the potential therapeutic effects of the miR-485 inhibitors disclosed herein on Alzheimer's disease, amyloid plaque formation and insoluble Aβ The effect of miR-485 inhibitors on levels was assessed in 10 month old 5XFAD mice. 5XFAD transgenic mice exhibit amyloid plaque deposition beginning at 2 months, and aggregation of Aβ in such plaques has been shown to exacerbate with progression of AD (Eimer et al., Mol Neurodegener 8 : 2 (2013); and Naslund et al., Proc Natl Acad Sci 91:8378-08382 (1994)).

Briefly, miR-485 inhibitors formulated with in vivo jetPEI reagent were injected into the right lateral ventricle of animals by stereotactic brain injection. Animals received a second dose one week later (see Figure 7A). Immunofluorescence microscopy with 6E10 staining and Thioflavin S was then used to quantify the number of amyloid plaque formations. As shown in FIGS. 7B and 7C, the number of amyloid plaques was significantly reduced in 5XFAD animals treated with miR-485 inhibitors compared to miR-control treated animals, indicating that miR-485 inhibitors It suggests that the amyloid burden in mice with AD can be ameliorated.

To further investigate the effects of miR-485 inhibitors on Aβ production, APP containing soluble Aβ1-42, amyloid precursor protein (APP), and α-secretase, ADAM (A disintegrin and metalloprotease) 10, and β-secretase BACE1 Levels of processing enzymes were assessed in the frontal cortex of AD mice in different treatment groups.

As shown in Figures 7D and 7E, soluble Aβ1-42 production was significantly reduced in AD mice treated with miR-485 inhibitors compared to control animals (ie, those treated with miR-control). miR-485 treated animals also showed decreased levels of β-CTF and sAPPβ (ie, the major product of BACE) in the frontal cortex compared to control animals (FIGS. 7F and 7G). Thus, AD animals treated with inhibitors also showed reduced expression of BACE1. Also supporting the results presented above (see Example 4), AD mice treated with miR-485 inhibitors showed significantly reduced levels of SIRT1 and PGC-1α proteins. However, some of the tested proteins were not negatively regulated by miR-485 administration. For example, there were no significant differences in total APP levels between animals in different treatment groups (see Figures 7F and 7G). In addition, administration of the miR-485 inhibitor significantly increased the expression of Adam10 and sAPPα proteins compared to control animals.

The above results demonstrate that the miR-485 inhibitors disclosed herein can modulate different genes, thereby reducing both Aβ production and plaque formation in vivo.

Example 8 Analysis of miR-485 Inhibitors on Phagocytosis of Aβ Plaques Alzheimer's disease is caused by an imbalance between Aβ production and clearance. Previous studies have shown that glial cells mediate clearance and phagocytosis of aggregated Aβ in AD brains and contribute to the alleviation of AD (Ries et al., Front Aging Neurosci 8:160 (2016). )). Therefore, to further investigate the role of glial cells in AD, immunohistochemical analysis using Iba1 and 6E10 antibodies was used to assess the co-localization of glial cells and Aβ plaques in mice with AD.

As shown in Figures 8A-8D, AD mice treated with miR-485 inhibitors showed significantly higher co-localization of Aβ plaques and glial cells. Furthermore, administration of miR-485 inhibitors to AD mice increased the uptake of Aβ plaques by primary glial cells (see Figure 8E).

To further assess phagocytosis and clearance of Aβ by glial cells, co-immunostaining for CD68, 6E10, and Iba1 was then used to quantify the number of CD68+ microglia phagosomes with internalized Aβ plaques. CD68 is one of the transmembrane glycoproteins of the lysosome/endosome-associated membrane glycoprotein family and functions as a scavenger receptor in debris clearance (Yamada et al., Cell Mol Life Sci 54(7):628- 40 (1998)).

As shown in FIGS. 8F and 8G, the clustering of Iba1+ microglia surrounding amyloid plaques increased in AD mice treated with miR-485 inhibitors compared to control animals (i.e., those treated with miR-control). A diffuse distribution of CD68 was shown.

To confirm the above results, Aβ aggregates were prepared by incubating Aβ monomers (100 μM) overnight at 37° C. followed by dilution of peptide stocks in cell culture medium. Primary glial cells were then transfected with miR-485 inhibitors and further treated with 1 μM fibrillar amyloid beta (fAβ) for 4 hours. Consistent with the above results, Aβ levels in conditioned media were significantly reduced in miR485-3p ASO-transfected cells compared to control-transfected cells (Fig. 8H).

The above results demonstrate that miR-485 inhibitors disclosed herein can enhance phagocytosis of Aβ by microglia.

Example 9 Analysis of CD36 Regulation by miR-485 Inhibitors As described herein, CD36/SR-BII can contribute to the phagocytosis of Aβ by glial cells. Using published algorithms (see Example 3), miR-485-3p was predicted to also have a binding site within the 3'UTR of CD36. Therefore, to assess whether the miR-485 inhibitors disclosed herein can also modulate CD36 expression, AD mice were treated with miR-485 inhibitors or miR-control (as described in the Examples above). The expression of CD36 in animals was evaluated after treatment with

As shown in Figures 9A and 9B, miR-485 inhibitor-treated AD mice showed significantly higher CD36 expression compared to control animals. Moreover, CD36 expression was significantly higher in Iba-1-positive microglial cells using immunohistochemistry (Fig. 9C).

Based on the above observations, it was next investigated whether transfection of miR485-3p or miR-485 inhibitors could alter the expression of CD36 in mouse primary glial cells. As shown in Figures 9D and 9E, CD36 expression was significantly reduced in miR485-3p-transfected primary glial cells compared to miR-controls. In contrast, miR-485 inhibitor transfected cells showed significantly higher CD36 expression.

The above results demonstrate that miR-485 inhibitors of the present disclosure can also increase CD36 expression by modulating miR-485-3p expression.

Example 10: Analysis of binding of miR485-3p to CD36 To confirm the target site for miR485-3p within the 3'UTR of CD36, luciferase reporter plasmids containing wild-type or mutant sequences of potential miR485-3p sites built. HEK293T cells were then transfected with these plasmids and promoter activity was measured in transfected cells. As shown in FIG. 10, in miR485-3p-transfected cells, wild-type promoter activity was significantly reduced, but the mutant did not differ.

Next, physical binding of miR485-3p to the 3'UTR of SIRT1 was assessed using an in vitro binding assay, as described in Example 5. Relative binding efficiency was significantly reduced in mutant seed sequences containing the 3'UTR.

Taken together, the above results indicate that miR485-3p can directly target the 3'UTR of CD36 and that this interaction can negatively regulate CD36 expression.

Example 11 Analysis of CD36 Regulation on Aβ Phagocytosis To further assess the role of CD36+ glial cells on Aβ phagocytosis, it was investigated whether CD36 inhibitory antibodies could affect glial cell phagocytosis. Briefly, primary glial cells were transfected with miR-485 inhibitor or miR-control. Transfected cells were treated with CD36 blocking antibody or control IgG followed by 1 μM fibrillar amyloid beta (fAβ) for 4 hours. An ELISA assay was used to examine Aβ phagocytosis in conditioned media harvested from different transfected cells.

As shown in FIG. 11, levels of Aβ were significantly reduced in miR-485 inhibitor-transfected cells compared to control-transfected cells. However, this effect was significantly reduced in cells treated with CD36 blocking antibody.

These results support that the miR-485 inhibitors disclosed herein can modulate CD36 expression in a miR485-3p-dependent manner, thereby affecting Aβ phagocytosis. is.

Example 12 Analysis of miR-485 Inhibitors on Neuroinflammation AD is known to be associated with inflammation in the brain, and the secretion of inflammatory mediators by fAβ-stimulated glial cells leads to neuronal loss and May contribute to cognitive decline (Cunningham et al., J Neurosci 25(40):9275-84 (2005)). Therefore, to assess whether the miR-485 inhibitors disclosed herein have any effect on neuroinflammation, primary glial cells were transfected with miR-485 inhibitors or miR-control followed by 1 μM of fibrillar amyloid beta (fAβ). Levels of intracellular SIRT1 and different inflammatory mediators (ie, NF-κB, TNF-α, and IL-1β) were then examined.

As shown in FIGS. 12A and 12B (and consistent with the data above (see Example 4)), SIRT1 expression was markedly decreased in primary glial cells treated with fAβ, whereas this The reduction was significantly restored in cells transfected with miR-485 inhibitors. Observed SIRT1 expression was correlated with NF-κB expression, as well as TNF-α and IL-1β expression levels (see FIGS. 12A and 12B). Cells transfected with miR-485 inhibitors showed a significant reduction in the levels of these inflammatory mediators, and this reduction appeared to be dose-dependent (see Figures 12I and 12J).

To further characterize the effects of miR-485 inhibitors on neuroinflammation, AD mice were treated with miR-485 inhibitors as described above (see Example 1). The expression patterns of Iba-1 (ie an activated microglial marker) and GFAP (ie an activated astrocyte marker) were then evaluated.

As shown in Figures 12C and 12D, microglia expressing high levels of Iba-1 and astrocytes expressing high levels of GFAP were significantly reduced in AD mice treated with miR-485 inhibitors. rice field. Expression levels of NF-κB, TNF-α, and IL-1β were also measured using real-time PCR, Western blot, and immunohistochemical analysis, as observed above in transfected cells. It was significantly lower in animals treated with miR-485 inhibitors (see Figures 12E-12H).

The above results demonstrate that the miR-485 inhibitors disclosed herein affect glial cell activation by reducing the expression of miR485-3p, through modulation of SIRT1/NF-κB signaling. This indicates that it can reduce the production of inflammatory cytokines.

Example 13 Analysis of miR-485 Inhibitors on Neuronal Loss and Postsynaptic Hyperplasia As noted above, 5XFAD transgenic mice exhibited amyloid plaque deposition beginning at 2 months and neuronal cell loss in the cortical layer at 9 months. (see Example 7). Synaptic and neuronal loss in 5XFAD mice has been correlated with Aβ accumulation and neuroinflammation (Eimer et al., Mol Neurodegener 8:2 (2013)). Given the results of the above examples (e.g., regulation of SIRT1 and CD36 expression by miR-485 inhibitors can control Aβ processing, phagocytosis, and inflammation in AD mice), disclosed herein We investigated whether the miR-485 inhibitors tested had any effect on neuronal cell death by assessing NeuN (a neuronal cell marker) and cleaved caspase-3.

Based on Western blot analysis, NeuN expression was increased while caspase-3 protein expression was decreased in cortical regions of miR-485 inhibitor-treated animals (see FIGS. 13A and 13B). However, this effect was not seen in the hippocampus under the same conditions. Similar results were observed using immunohistochemical analysis (see Figures 13C and 13D).

Next, the effect of miR-485 inhibitors on postsynaptic hyperplasia was examined by assessing PSD-95 expression. As shown in Figures 13E and 13F, PSD-95 protein expression was significantly higher in the frontal cortex of AD mice treated with miR-485 inhibitors compared to control animals.

The above results demonstrate that the inhibitors of miR-485 disclosed herein can not only minimize neuronal cell loss, but also increase postsynaptic hyperplasia. It further demonstrates the therapeutic effect.

Example 14 Analysis of miR-485 Inhibitors on Cognitive Function Whether the results observed above in Example 13 (ie, increased postsynaptic hyperplasia and decreased neuronal loss) correlate with improved cognitive function. To examine , AD mice were again treated with miR-485 inhibitors or miR-control as described in the Examples above. The animals' cognitive function was then assessed using the Y-maze and the Passive Avoidance Test (PAT), a widely accepted behavioral paradigm for assessing spatial working memory.

We found that mice treated with miR-485 inhibitors had significantly increased spontaneous alternation rate (%) 2 days after the last injection. Total arm entries were not significantly different between control and miR-485 inhibitor-treated 5XFAD, indicating that levels of general motor and exploratory behavior in the Y-maze were unchanged ( Figure 14A). In addition, we investigated associative memory in passive avoidance tests based on associations formed between electrical paw stimulation and a specific environmental context of spontaneous preference (dark vs. light). Migration latencies were similar in controls and 5XFAD treated with miR-485 inhibitors. However, mice treated with miR-485 inhibitors showed a significant reduction in latency to stay in the dark compartment 24 hours after being electroshocked (Fig. 14B).

Taken together, the above results demonstrate that the miR-485 inhibitors disclosed herein can modulate (i.e., increase) the expression of different genes involved in neurodegenerative diseases such as AD. . As shown in the Examples above, these genes include SIRT1, CD36, and PGC-1α. While not wishing to be bound by any one theory, the above results suggest that the miR-485 inhibitors disclosed herein regulate many aspects of AD by modulating the expression of these genes. (e.g., reduce both Aβ production and plaque formation, promote phagocytosis of Aβ plaques, reduce neuroinflammation, increase postsynaptic hyperplasia, and improve cognitive function) (Fig. 15).

Example 15: Analysis of efficacy of miR-485 inhibitors in modulating SIRT1, PGC-1α, and CD36 expression in vivo Disclosed herein in modulating SIRT1, PGC-1α, and CD36 expression To further evaluate the efficacy of miR-485 inhibitors, a single dose (100 μg/mouse; 5 mg/kg) of miR-485 inhibitors (see Examples) was administered to wild-type males purchased from KOATECH (Korea). Crl:CD1 (ICR) mice were administered (by intravenous administration). Control animals received miR-control (see Example 1). Animals were then sacrificed at different time points after dosing and Western blots were used to assess the expression levels of SIRT1, PGC-1α and CD36 in both brain cortex and hippocampus.

As shown in Figures 16A-16C, Figures 17A-17C, and Figures 18A-18B, a single administration of miR-485 inhibitors resulted in rapid expression of SIRT1, PGC-1α, and CD36 in both cortex and hippocampus. increased to For SIRT1, peak expression was observed in the cortex approximately 48 hours after administration (approximately 300% increase over expression in control animals) and in the hippocampus approximately 24 hours after administration (approximately 150% increase relative to controls). % increase) (see Figures 16A and 17A, respectively). A peak expression of PGC-1α was also observed in the cortex approximately 48 hours after administration (approximately 100% increase over controls) and in the hippocampus approximately 24 hours after administration (approximately 50% increase over controls). increase) (see Figures 16B and 17B, respectively). Similar results were observed for CD36 (see Figure 18A).

These results support the efficacy of miR-485 inhibitors disclosed herein in modulating the expression of SIRT1, PGC-1α, and CD36. In comparison, the small molecule ApoE4 has been shown to have a positive effect in normalizing SIRT1 expression in vivo (Campagna et al., Sci Rep 8(1):17574 (Dec. 2018). )). After 56 days of daily dosing (dose of 40 mg/kg per day), there was an approximately 20% increase in SIRT1 expression in the hippocampus, but not in the cortex. With a miR-485 inhibitor disclosed herein (eg, SEQ ID NO: 28), a single administration of a significantly lower dose (i.e., 5 mg/kg) significantly elevated SIRT1 in both hippocampus and cortex. resulted in the expression of

Example 16 Analysis of Therapeutic Effect of miR-485 Inhibitors on Parkinson's Disease In addition to the examples presented above, the therapeutic effects of miR-485 inhibitors disclosed herein on Parkinson's disease can be analyzed, for example, in the present invention. Thiele et al., the disclosure of which is incorporated herein by reference in its entirety. , J Vis Exp. 60:3234 (2012) using the 6-OHDA mouse model. Specifically, the effects of miR-485 inhibitors on dopaminergic degeneration were assessed. A Unilateral 6-OHDA Model Induces Partial Striatal Lesion with Progressive Retrograde Nigrostriatal Pathology and Allows Evaluation Based on Behavioral and Neurochemical Parameters Associated with PD It is a thing.

Briefly, mice were injected intraperitoneally with desipramine (25 mg/kg in 0.9% NaCl) approximately 30 minutes prior to administration of 6-hydroxydopamine (6-OHDA), followed by isotroy vapor (Toikaa Pharmaceuticals Limited). , Gujarat, India). Anesthetized mice were placed in a brain stereotaxic frame (JEUNG DO BIO & PLANT CO., LTD, Seoul, Korea) and positioned in the right striatum (anterior-posterior to bregma: +0.9 mm; medial-lateral: -2.0 mm). dorsoventral: −2.5 mm) at a rate of 0.5 μl/min for a total dose of 15 μg/3 μl of 6-OHDA (5 μg/μl in 0.02% ascorbic acid dissolved in 0.9% NaCl). Sigma Aldrich) was administered by unilateral injection. All injections were performed using a Hamilton syringe (30S needle) attached to a syringe pump (Harvard Apparatus, Holliston, Mass., USA). After injection, the needle was slowly withdrawn 5 minutes later.

One day after administration of 6-OHDA to induce brain lesions in animals, animals were treated with either a miR-485 inhibitor (50 μg/head) (SEQ ID NO:30) or a control oligonucleotide (SEQ ID NO:61) alone. One dose was administered by intravenous administration (tail vein injection) (see Figure 23A). Six days after miR-485 inhibitor administration, the animals' motor function was assessed using one or more of the following tests: pole test, rotarod, hang wire test, and balance beam (performed See Example 1). Animals were sacrificed 9 days after miR-485 inhibitor administration and the effects of miR-485 inhibitor administration on brain tissue were assessed by measuring tyrosine hydroxylase expression using Western blots. The effect of miR-485 inhibitors on neuroinflammation was also assessed using Western blot analysis.

As shown in Figures 23B-23E, 6-OHDA mice treated with miR-485 inhibitors showed at least rotarod (showed increased latency to fall time), hang wire test (latency to fall time), showed an increase in time), and an improvement in motor function as measured using the balance beam (fewer foot slides). Animals treated with miR-485 inhibitors also showed higher expression of tyrosine hydroxylase (i.e., a marker of dopaminergic neurons) in both the substantia nigra (SN) and striatum (STR), indicating that dopaminergic neurons (see Figures 23F-23I). A significant decrease in IL-1β expression was observed in the substantia nigra of miR-485 inhibitor-treated animals compared to control animals (see Figures 23J and 23K). However, administration of miR-485 inhibitors did not appear to significantly affect the expression of Iba-1 (activated microglial marker), GFAP (activated astrocyte marker), and TNF-α.

Next, to further evaluate the therapeutic effects of miR-485 inhibitors on Parkinson's disease, additional 6-OHDA mice were treated with the following doses: 2.5 mg/kg or 5 mg/kg of either miR-485 inhibitor. was treated by a single intravenous administration of Healthy 6-OHDA mice treated with PBS or control oligonucleotides were used as controls. Six days after miR-485 inhibitor administration, the animals' motor function was then assessed using one or more of the following tests: pole test, rotarod, hang wire test, and balance beam. (See Example 1).

As shown in Figures 39A-39D, consistent with previous data, miR-485 inhibitor-treated animals showed significant improvement in motor function at both doses. Taken together, the results presented in this example demonstrate that miR-485 inhibitors disclosed herein can ameliorate neuronal damage associated with diseases such as Parkinson's disease (e.g., dopaminergic neuronal damage). reduced injury and/or reduced neuroinflammation), suggesting that this may improve motor function.

Example 17 Analysis of the Effects of miR-485 Inhibitors on Autophagy As described herein, autophagy plays an important role in maintaining cellular homeostasis by regulating long-lived proteins, protein aggregates, and injury. It plays an important role in the proper degradation of organelles. Therefore, to assess whether the miR-485 inhibitors disclosed herein have any effect on autophagy in cells of the CNS, primary cortical neurons and primary mixed glial cells were treated with different concentrations of miR-485. A combination of inhibitors and mouse α-synuclein PFF (aggregated) (mPFF) (1 μg/mL) was treated for 24 or 48 hours. Expression levels of intracellular p62 (an adapter molecule that recruits substrates to autophagosomes) and LC3B (a marker of autophagosome formation) were then assessed using Western blot analysis.

As shown in Figures 24A and 24B, miR-485 inhibitors at all concentrations tested had no significant effect on p62 expression in primary cortical neurons. However, treatment with miR-485 inhibitors resulted in significant restoration of LC3B expression in mPFF-treated primary cortical neurons. For example, after 48 hours, minimal LC3B expression was detected in primary cortical neurons treated with mPFF alone (see FIG. 24A, second vertical lane of right gel). However, LC3B expression increased progressively with increasing concentrations of miR-485 inhibitor (see FIG. 24A, 3rd, 4th, and 5th vertical lanes of right gel). Similar results were observed in primary mixed glial cells (see Figure 24B).

To confirm the above results, BV2 microglial cells were transfected with different doses of miR-485 inhibitors (0 nM, 50 nM, 100 nM, and 300 nM) followed by fibrillar amyloid beta (oAβ) for 24 hours at a final concentration of Treated at 1 μM. Expression levels of different proteins associated with autophagy, namely FOXO3a, LC3 and p62, were then assessed using Western blot analysis. As shown in Figures 40A-40D, dose-dependent increases in the expression levels of these proteins were observed in cells transfected with miR-485 inhibitors.

Taken together, the above results demonstrate that the miR-485 inhibitors disclosed herein can increase autophagic flux in both primary cortical neurons and glial cells, and the neurodegeneration disclosed herein. It has been shown to be useful in treating diseases such as Parkinson's disease and/or Alzheimer's disease.

Example 18 Analysis of Safety Profiles of miR-485 Inhibitors A single dose toxicity study was conducted to assess whether in vivo administration of miR-485 inhibitors could result in any adverse effects. Briefly, miR-485 inhibitors were administered at the following doses: (i) 0 mg/kg (G1), (ii) 3.75 mg/kg (G2), (iii) 7.5 mg/kg (G3), and (iv) administered to male and female rats at one of 15 mg/kg (G4). Body weight, mortality, clinical signs, and pathological abnormalities were then observed in animals at different time points after transfer.

As shown in Figures 19A and 19B, administration of miR-485 inhibitors (at all doses tested) did not appear to have any abnormal effects on body weight in both male and female rats. Similarly, no mortality or pathological abnormalities were observed in any of the treated animals (see Figures 20A, 20B, 22A and 22B). With regard to possible clinically relevant side effects (e.g., NOA, congestion (tail), and edema (face, forelimbs, or hindlimbs)), such effects disappeared by 1 day post-dosing in all treated animals. (See Figures 21A and 21B).

Taken together, the above results demonstrate that the miR-485 inhibitors disclosed herein are not only effective in modulating the expression of SIRT1, PGC-1α, and CD36, but are also safe when administered in vivo. It shows that

Example 19 Development of a New Alzheimer's Disease Animal Model by Viral Delivery System of miRNA A new animal model was developed to further evaluate the role. In summary, one or more of the following materials and methods were used in constructing new AD animal models.

Lentiviral Transfer Plasmid The sequence of the pLenti-III-mir-GFP vector (for example, 485-3p-lenti-mini-7-GFP-F) containing the mature mouse miR-485-3p is as follows (miR- 485-3p sequences are shown in upper case):
Sequence of the Lenti-mir-GFP-cloning vector:
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Lentiviral Production in 293T Cells HEK293T cells were seeded in 10 cm tissue culture plates until reaching 70-80% confluence (eg 3×10 ⁶ cells in 10 ml of DMEM complete medium). Two hours before viral DNA transfection, the medium was removed from the 293T cells and replaced with 5 ml of DMEM medium. TransIT®-Lenti reagent (Mirus Bio, Catalog Nos. 6600, 6603, 6604, 6605, 6606, and 6610) was warmed to room temperature and gently vortexed. 1 ml of Opti-MEM reduced serum medium was placed in a sterile tube. 10 ug of pMDLg/pRRE packaging plasmid (Addgene, Plasmid #12251), 5 ug of pRSV-REV packaging plasmid (Addgene, Plasmid #12253), 2.5 ug of pMD2. G envelope plasmid (Addgene, Plasmid #12259) and 2.5 ug of 485-3p-lenti-mini-7-GFP-F plasmid (as described above) were combined in another sterile tube and mixed well. , were transferred to tubes containing reduced serum Opti-MEM medium. 35 ul of TransIT®-Lenti reagent was added to the dilution and incubated for 20 minutes at room temperature to form transfection complexes. TransIT®-Lenti reagent:DNA complexes (prepared above) were spread dropwise onto 10 cm culture plates containing 293T cells. Lentivirus was harvested after incubating the transfected cell cultures at 37° C. in 5% CO 2 for 72 hours.

After 72 hours of incubation, cells were centrifuged at 6000 rpm for 15 minutes in sterile tubes. The virus-containing supernatant was ultracentrifuged at 29000 rpm for 2 hours to obtain a virus-containing pellet. The pellet was diluted 1:1000 in cold PBS and stored at -80°C.

Viral Titration To establish titers of virus preparations, 293T cells were seeded at 4×10 ⁵ cells per well in 6-well tissue culture plates in complete DMEM medium. The following day, approximately 1 mL of media was left in each well and 1 μL of lentiviral construct (either control vector or vector containing miR-485-3p and GFP) and polybrene (8 μg/mL) were added to each well. The lentiviral constructs were serially diluted (1, 0.1, and 0.01) prior to addition. After 6 hours of incubation, the medium was replaced with fresh medium. After 48 hours, cells were washed with warm PBS and analyzed for GFP expression by flow cytometry (BD Accuri C6 plus).

Animals Mice (wild type, male, C57BL/6J, 6 weeks old) were purchased from Dae Han Bio Link Co Ltd (Chungju-si, Republic of Korea). Mice undergoing surgery and behavioral experiments were housed in one cage to eliminate physical injury and psychological anxiety caused by aggression from other males. Water and food were available ad libitum in a 12 hour light/12 hour dark cycle environment.

Both lentiviral control vector (without miR-485-3p) and mice injected into the brain with lenti-miR-485-3p vector were routinely checked for any physical abnormalities in postoperative appearance and weight. All behavioral experiments were performed during the light period and all mice in each group were tested under the same conditions.

In Vivo Lentiviral Vector Injection and Tissue Preparation Six-week-old mice were treated with anesthesia (2,2,2-tribromoethanol (250 mg/kg ip, Sigma-Aldrich, Cat. No. 75-80-9))). was anesthetized by intraperitoneal injection using Using a stereotaxic surgical device and a Hamilton syringe 700 series, the lentiviral vector was injected into the dentate gyrus and CA1 region of the hippocampus (AP = -2 mm, L = ±1.5 mm, ventral (V) = -2.7). and −2.0 mm). The virus volume per site was 1.5 ul, the injection flow rate was 0.2 ul/min, and the remaining time after injection was 15 minutes. After surgery, the process of recovery from anesthesia and body weight were checked to determine if there were any health problems resulting from the surgery.

After completion of behavioral experiments, mice were anesthetized and sacrificed by cardiac perfusion, brains were carefully removed and post-fixed in 4% paraformaldehyde at 4°C for 4 hours followed by 30% sucrose/0. Cryopreserved in 1 M PBS for approximately 48 hours. Brains were embedded in OCT compound (Tissue-Tek®, Sakura, Inc., Catalog No. 4583) and sectioned into 40 μm thick sections at −22° C. using a Leica CM1860 cryostat (Leica Microsystems). It was sectioned longitudinally. For viral GFP images, nuclei were stained with DAPI (1:500; Invitrogen, cat#D3571) and mounted using fluorescent antifade mounting medium. Images were acquired using a confocal microscope (Leica DMi8).

Behavioral Tests Mouse behavioral tests were performed in the following order. (i) open field test, (ii) Y-maze, (iii) novel object recognition test, and (iv) passive avoidance test (see, eg, FIG. 26). Upon completion of one behavioral experiment, a 2-day recovery period was allowed before the next experiment. Behavioral experiments, excluding passive avoidance, were analyzed using the smart 3.0 video tracking system (Panlab, worldwideweb.harvardapparatus.com/smart-video-tracking-system).

(i) Open field test Mice were placed in the center of a white mat chamber (450 mm x 450 mm x 450 mm) and allowed to move freely for 30 minutes. Digital video tracking was performed. Basal movement was measured during 30 minutes by analyzing total distance in cm steps, recording central distance (distance traveled within the central compartment) (cm) and total distance traveled across the area (cm). Central compartment activity (%) was calculated by dividing the central distance by the total distance and multiplying by 100. The ratio of center distance:total distance can be used as an index of anxiety-related responses.

(ii) Y-maze The Y-maze consisted of three white matt plastic arms (65 mm x 400 mm x 130 mm) angled at 120° to each other. Mice were centered and allowed to move freely for 8 min, exploring all three arms. The number of arm entries and trials (alternating behavior is entry into three separate arms 10 cm from the center) was recorded and the alternation behavior rate was calculated. Entry was defined as all three limbs entering the arms of the Y-maze. The alternation rate was defined as the number of trials divided by the number of arm entries −2 multiplied by 100.

(iii) Novel Object Recognition Test Mice were placed in the center of a white mat chamber (450 mm×450 mm×450 mm) and allowed to move freely for 5 min on the first day (Day 1) to adapt to the space. Twenty-four hours later (day 2), two identical objects were placed in the first and fourth compartments of the chamber and the mice were allowed to move freely for 10 minutes to interact with the two objects (A and A) and space. I learned about One hour after measuring short-term memory, one of the two objects was replaced with one of a different shape and color (A and B) and curiosity (number of nose poking) to the novel object was measured. To measure long-term memory, after 24 hours (day 3) and 3 weeks (day 24), one of the two objects is replaced with a different shape and color (e.g., from A and C to A and D). 2), measured and analyzed the number of nose-pokes of novel objects. Results were compared by analyzing the number of nose pokes for novel and familiar objects for each group, and a discrimination index was calculated by the following formula. [(novel object - familiar object) / (novel object + familiar object)]

(iv) Passive Avoidance Test A passive avoidance test was performed as described in Example 1.
Cell Culture Cultivation of primary mouse neurons

For example, Seibenhener ML. et al. , J Vis Exp. , (65):3634 (2012), hippocampal-containing cortices were isolated from the heads of 18.5-day-old fetal mouse mice and subjected to primary culture. On day 8 of DIV (days in vitro), neurons were transfected with 485-3p-lenti-mini-7-GFP-F lentiviral vector or lentiviral control vector (without miR-485-3p) using polybrene. ), and GFP expression was observed 30 hours later. After confirming that cells were properly infected, immunohistochemistry was performed after fixation using 4% paraformaldehyde as described above (after 30 hours) (e.g., In Vivo Lentiviral Vector Injections and Tissue Preparation).

Cultivation of Mouse Primary Glial Cells One-day-old mice were sacrificed and mixed glial cells were cultured. For example, Lian H. , et al. , Bio Protoc. , 6(21):e1989 (2016), microglial cells were isolated on DIV (days in vitro) day 10 and astrocytes were isolated on DIV day 11 to establish glial cell cultures. prepared. On DIV day 14, glial cells were transduced with 485-3p-lenti-mini-7-GFP-F lentiviral vector or lentiviral control vector (without miR-485-3p) using polybrene and incubated for 30 hours. GFP expression was observed later. After confirming that the cells were properly infected, immunohistochemistry was performed after fixation using 4% paraformaldehyde as described above (see, e.g., In Vivo Lentviral Vector Injections and Tissue Preparation). .

485-3p-lenti-mini-7-GFP-F lentiviral vector in human microglial primary cells from the central nervous system (CNS) cortex, immortalized human astrocytes, and fetal SV40 cells using the human cell line polybrene. Alternatively, cells were transduced with a lentiviral control vector (without miR-485-3p) and GFP expression was observed 24 hours later. After confirming that the cells were properly infected, immunohistochemistry was performed after fixation using 4% paraformaldehyde as described above (see, e.g., In Vivo Lentiviral Vector Injections and Tissue Preparation). .

Disease Induction CA1 is known to be a part of the brain that plays an important role in the development of AD. The pathology of AD is thought to start from distal CA1, which is the boundary between hippocampal CA1 and subiculum, and progress to CA2 (for example, Arjun V. Masurkar, J Alzheimers Dis Parkinsonism, 8 (1) : 412 (2018)). Therefore, both regions were selected as target sites for overexpression of miR-485-3p to inhibit neurogenesis in the dentate gyrus of the hippocampus and induce pathology in CA1.

The neural circuit of the rodent hippocampus is composed of an excitatory trisynaptic circuit (entorhinal cortex (EC)-dentate gyrus (DG)-CA3-CA1-EC) (see, eg, Wei D. et al. , Nat Rev Neurosci, 11:339-350 (2010)). The experiment was designed to project to the entire hippocampal region, including CA3-CA1. There appears to be a limitation in expressing miR-485-3p throughout the hippocampus when the lentiviral vector is injected by choosing only one coordinate. Therefore, injection coordinates were set so that lentivirus injection into the posterior hippocampus would project virus-infected neurons to the anterior hippocampus.

DG and CA1 of mouse hippocampus were injected with vector containing lenti-miR485-3p GFP-F at 2 points per hemisphere (4 points in total) (Fig. 25A). As shown in FIG. 25B, significant GFP expression was observed in the dentate gyrus and CA1 of both the pre- and post-hippocampus. This result indicates that miR-485-3p was extensively and effectively overexpressed throughout the mouse hippocampus using the method described above.

Example 20 Observation of Behavioral Changes Related to Cognitive Function by Overexpression of miR485-3p Next, the effects of miR485-3p overexpression on behavioral changes related to cognitive function, learning, and memory were observed. In summary, approximately one month after miR485-3p overexpression, the different behavioral tests described in Example 19 were performed as described in FIG.

Open Field Test According to the open field test (which measures changes in basal locomotion and anxiety-related responses), locomotion (total distance traveled) was significantly increased in the group of mice injected with the lenti-miR485-3p vector compared to the control group. No difference was seen (Fig. 27A). Similar results were obtained when central zone activity (i.e., length of time spent in the central portion of the open field arena; increased time in the central area was considered indicative of anxiety-like activity) between the two groups. was observed (Fig. 27B). Thus, overexpression of miR485-3p in mouse hippocampus did not affect motor and emotion-related (eg, anxiety) functions.

Y-maze Test As described herein (eg, Example 14 and Example 19), the Y-maze test is a behavioral experiment that assesses spatial working memory. This test is based on the finding that normal rodents prefer to explore novel environments (e.g., normal mice move from previously visited arm to previously visited arm of the Y-shaped apparatus). I generally prefer exploring new arms to returning to ). Various brain regions are involved in performing this action, including the hippocampus, septum, basal forebrain, and prefrontal cortex. Therefore, the Y-maze test can be useful in assessing the proper functioning of any of these different brain regions.

As observed, there was no statistically significant difference in the total number of arm entries between control and miR485-3p overexpression groups (Fig. 28A). Similarly, alternation behavior (ie, the finding that normal mice chose a different arm than the arm from which they came) was comparable with different animal sensations (Fig. 28B). These results indicate that the level of general locomotion and exploratory behavior in the Y-maze did not change after overexpression of miR-485.

Novel Object Recognition Test As described herein (e.g., Example 19), the Novel Object Recognition Test is a behavioral test often used in rodent models to assess potential defects in object recognition memory. is. This test is based on the property of mice to explore novel objects with greater curiosity than familiar objects, and measures both short-term and long-term memory.

Compared to the control group (i.e., without miR485-3p overexpression in the hippocampus), the mice in the experimental group (i.e., those with overexpression of miR485-3p in the hippocampus) had a short-term (object recognition training) 29B and 29E) and long-term (24 hours after object recognition training (see FIGS. 29C and 29F) and 3 weeks (FIGS. 29D and 29G)). showed a statistically significant impairment in performance. Animals in the control group were able to distinguish between familiar and novel objects and showed greater interest in exploring novel objects. In contrast, mice overexpressing miR485-3p did not distinguish between familiar and novel objects. These results suggest that overexpression of miR485-3p in the hippocampus has an adverse effect on both short-term and long-term memory formation.

Passive Avoidance Test As described herein, the passive avoidance test is a fear-motivated test. This test tests the ability of mice to perceive and learn environments (ie, associative memory) to avoid environments in which an aversive stimulus such as a foot shock is presented.

As shown in FIG. 30, there was no significant difference in entry latency values between the control group and the miR485-3p overexpression group. This data indicates that overexpression of miR485-3p in mouse hippocampus did not significantly affect fear acquisition.

Taken together, the above results demonstrate that miR-485 overexpression in the hippocampus impairs both short-term and long-term memory, as observed in many AD patients. The above results demonstrate that miR-485 inhibitors disclosed herein may be useful in improving certain cognitive functions (e.g., short-term and long-term memory) in AD patients (e.g., in different brain regions). by reducing the expression of miR-485).

Example 21: Effect of overexpression of miR485-3p on neurons To observe whether it was induced, neurons were transduced with lenti-miR485-3p (experimental group) or lenti-control vector (control group) as described in Example 19 (see Figure 31A).

FIG. 31B shows increased and accumulated amyloid beta in cells that overexpressed miR-485-3p relative to the lenti-control group. Amyloid-beta was also observed in cells not infected with virus in the experimental group, indicating that amyloid-beta spreads from neuron to neuron. A truncated tau protein, known to be a neuropathological hallmark of AD, was also observed to be increased in the miR485-3p overexpression group compared to the lenti-control group (Fig. 32). Furthermore, in neurons transduced to overexpress miR485-3p, PSD-95 (a key scaffolding protein that regulates synaptic distribution and activity of both NMDA and AMPA receptors; see Figure 33A) and synaptophysin (which may play a role in the release of neurotransmitters from synaptic vesicles by regulating premembrane fusion, see Figure 33B). Without wishing to be bound by any one theory, in some aspects, miR485-3p overexpression increases neuronal amyloid beta expression, induces tauopathy, impairs synaptogenesis, and/or Alternatively, it may adversely affect neuronal cell fate by increasing neuronal cell death (see Figure 34).

Next, to assess whether overexpression of miR485-3p could also affect other neurons, mouse primary astrocytes and microglial cells were infected with lenti-miR485-3p as described in Example 19. or transduced with a lenti-control vector (see Figure 35A). Lentiviral infection resulted in the expression of Iba-1, a cell-specific marker for microglia isolated from mouse whole brain (Fig. 35B) and GFAP, a cell-specific marker for astrocytes (Fig. 36A), and each cell This was confirmed by observing the characteristics of the GFP signal. In both microglia (FIGS. 35C and 37C) and astrocytes (FIGS. 36B and 38B), cleaved caspase-3 was increased as a result of overexpression of miR485-3p. These results indicate that miR485-3p not only directly damages neurons, but also induces glial cell gliosis, which plays an important role in neuroinflammation and synaptic homeostasis.

The results described above in Examples 19-21 further demonstrate the role that miR485-3p expression may have in the induction of Alzheimer's disease. While not wishing to be bound by any one theory, miR-485 inhibitors described herein may reduce and/or inhibit miR485-3p expression, resulting in various neurological disorders such as Alzheimer's disease. It can be a useful therapeutic agent in the treatment of degenerative diseases and disorders.

It should be appreciated that the "Detailed Description of the Invention" section is intended to be used in interpreting the claims, rather than the "Summary" and "Abstract" sections. The Summary and Summary sections may present one or more, but not all, exemplary aspects of the present disclosure contemplated by the inventor(s), and thus the scope of the present disclosure and the appended claims. is not intended to limit in any way.

The disclosure has been described above using functional building blocks to denote the performance of specific functions and relationships thereof. The boundaries of these functional elements have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are properly performed.

Because the above specific descriptions of specific embodiments are fully indicative of the general nature of this disclosure, others may apply knowledge within the skill of those in the art to avoid undue experimentation. Such specific aspects may be readily modified and/or adapted to various uses without modification and without departing from the general concepts of this disclosure. Therefore, such adaptations and modifications are intended to be encompassed within the meaning and range of equivalents of the disclosed aspects, based on the teaching and advice presented herein. The phrases and terms used herein are for the purpose of description and not of limitation, and the phrases or terms used herein may be understood by one of ordinary skill in the art after considering the teachings and advice herein. should be understood by

The breadth and scope of the present disclosure should not be limited by any of the exemplary aspects described above, but should be defined only in accordance with the following claims and their equivalents.

The entire contents of all cited references (including references, patents, patent applications, and websites) that may be cited throughout this application are expressly incorporated herein by reference for all purposes, and , the literature cited therein is likewise incorporated.

Claims (110)

A method for increasing the level of SIRT1 protein and/or SIRT1 gene in a subject in need of increasing the level of SIRT1 protein and/or SIRT1 gene, comprising administering a compound that inhibits miR-485 (miRNA inhibitor) to the subject The above method, comprising administering. 2. The method of claim 1, wherein the subject has a disease or condition associated with decreased levels of SIRT1 protein and/or SIRT1 gene. 3. The method of claim 1 or 2, wherein the miRNA inhibitor induces autophagy and/or treats or prevents inflammation. A method of increasing CD36 protein and/or CD36 gene levels in a subject in need thereof, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor) to said subject The above method, comprising administering. 5. The method of any one of claims 1-4, wherein the subject has a disease or condition associated with decreased levels of CD36 protein and/or CD36 gene. A method of increasing the level of PGC-1α protein and/or gene of PGC-1α in a subject in need of increasing the level of PGC-1α protein and/or gene of PGC-1α comprising a compound that inhibits miR-485 ( administering a miRNA inhibitor) to the subject. The method of any one of claims 1-6, wherein the subject has a disease or condition associated with decreased levels of PGC-1α protein and/or PGC-1α gene. A method for increasing the level of the LRRK2 protein and/or the LRRK2 gene in a subject in need of increasing the level of the LRRK2 protein and/or the LRRK2 gene, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor) to the subject The above method, comprising administering. The method of any one of claims 1-8, wherein the subject has a disease or condition associated with decreased levels of LRRK2 protein and/or LRRK2 gene. A method for increasing NRG1 protein and/or NRG1 gene levels in a subject in need thereof, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor) to said subject The above method, comprising administering. The method of any one of claims 1-10, wherein the subject has a disease or condition associated with decreased levels of NRG1 protein and/or NRG1 gene. A method for increasing STMN2 protein and/or STMN2 gene levels in a subject in need thereof, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor) to said subject The above method, comprising administering. 13. The method of any one of claims 1-12, wherein the subject has a disease or condition associated with decreased levels of the STMN2 protein and/or the STMN2 gene. A method for increasing VLDLR protein and/or VLDLR gene levels in a subject in need thereof, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor) to said subject. The above method, comprising administering. 15. The method of any one of claims 1-14, wherein the subject has a disease or condition associated with decreased levels of VLDLR protein and/or VLDLR gene. A method for increasing NRXN1 protein and/or NRXN1 gene levels in a subject in need of increasing NRXN1 protein and/or NRXN1 gene levels, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor) to said subject The above method, comprising administering. 17. The method of any one of claims 1-16, wherein the subject has a disease or condition associated with decreased levels of NRXN1 protein and/or NRXN1 gene. A method for increasing the level of GRIA4 protein and/or GRIA4 gene in a subject in need of increasing the level of GRIA4 protein and/or GRIA4 gene, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor) to said subject The above method, comprising administering. 19. The method of any one of claims 1-18, wherein the subject has a disease or condition associated with decreased levels of GRIA4 protein and/or GRIA4 gene. A method for increasing the level of NXPH1 protein and/or NXPH1 gene in a subject in need of increasing the level of NXPH1 protein and/or gene, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor) to said subject The above method, comprising administering. 21. The method of any one of claims 1-20, wherein the subject has a disease or condition associated with decreased levels of NXPH1 protein and/or NXPH1 gene. A method of increasing PSD-95 protein and/or PSD-95 gene levels in a subject in need thereof, comprising a compound that inhibits miR-485 ( administering a miRNA inhibitor) to the subject. 23. The method of any one of claims 1-22, wherein the subject has a disease or condition associated with decreased levels of PSD-95 protein and/or PSD-95 gene. A method for increasing the level of synaptophysin protein and/or synaptophysin gene in a subject in need thereof, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor) to said subject The above method, comprising administering. 25. The method of any one of claims 1-24, wherein the subject has a disease or condition associated with decreased levels of synaptophysin protein and/or synaptophysin gene. A method of reducing caspase-3 protein and/or caspase-3 gene levels in a subject in need thereof, comprising a compound that inhibits miR-485 ( administering a miRNA inhibitor) to the subject. 27. The method of any one of claims 1-26, wherein the subject has a disease or condition associated with increased levels of caspase-3 protein and/or caspase-3 gene. 28. The method of any one of claims 1-27, wherein the miRNA inhibitor induces neurogenesis. 29. The method of claim 28, wherein inducing neurogenesis comprises increasing proliferation, differentiation, migration and/or survival of neural stem and/or progenitor cells. 30. The method of claim 28 or 29, wherein inducing neurogenesis comprises increasing the number of neural stem and/or progenitor cells. 30. The method of claim 28 or 29, wherein inducing neurogenesis comprises increasing axonal, dendrite and/or synaptic development. 32. The method of any one of claims 1-31, wherein the miRNA inhibitor induces phagocytosis. A method of treating a disease or condition associated with aberrant levels of the SIRT1 protein and/or SIRT1 gene in a subject in need thereof, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor). The above method, comprising administering to the subject, wherein the miRNA inhibitor increases the level of the SIRT1 protein and/or SIRT1 gene. A method of treating a disease or condition associated with abnormal levels of the CD36 protein and/or CD36 gene in a subject in need thereof, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor). The above method, comprising administering to the subject, wherein the miRNA inhibitor increases the level of the CD36 protein and/or CD36 gene. A method of treating a disease or condition associated with aberrant levels of the PGC-1α protein and/or PGC-1α gene in a subject in need thereof, comprising a compound that inhibits miR-485 (miRNA inhibitor) to said subject, wherein said miRNA inhibitor increases the level of said PGC-1α protein and/or PGC-1α gene. A method of treating a disease or condition associated with aberrant levels of the LRRK2 protein and/or LRRK2 gene in a subject in need thereof, comprising a compound that inhibits miR-485 (a miRNA inhibitor). The above method, comprising administering to the subject, wherein the miRNA inhibitor increases the level of the LRRK2 protein and/or LRRK2 gene. A method of treating a disease or condition associated with aberrant levels of the NRG1 protein and/or NRG1 gene in a subject in need thereof, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor). The above method, comprising administering to the subject, wherein the miRNA inhibitor increases the level of the NRG1 protein and/or NRG1 gene. A method of treating a disease or condition associated with aberrant levels of the STMN2 protein and/or the STMN2 gene in a subject in need thereof, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor). The method, comprising administering to the subject, wherein the miRNA inhibitor increases the level of the STMN2 protein and/or STMN2 gene. A method of treating a disease or condition associated with abnormal levels of a VLDLR protein and/or VLDLR gene in a subject in need of treatment, comprising a compound that inhibits miR-485 (a miRNA inhibitor). The above method, comprising administering to the subject, wherein the miRNA inhibitor increases the level of the VLDLR protein and/or VLDLR gene. A method of treating a disease or condition associated with aberrant levels of the NRXN1 protein and/or NRXN1 gene in a subject in need thereof, comprising a compound that inhibits miR-485 (a miRNA inhibitor). The method, comprising administering to the subject, wherein the miRNA inhibitor increases the level of the NRXN1 protein and/or NRXN1 gene. A method of treating a disease or condition associated with aberrant levels of the GRIA4 protein and/or GRIA4 gene in a subject in need thereof, comprising a compound that inhibits miR-485 (a miRNA inhibitor). The above method, comprising administering to the subject, wherein the miRNA inhibitor increases levels of the GRIA4 protein and/or GRIA4 gene. A method of treating a disease or condition associated with aberrant levels of the NXPH1 protein and/or NXPH1 gene in a subject in need thereof, comprising administering a compound that inhibits miR-485 (a miRNA inhibitor). The above method, comprising administering to the subject, wherein the miRNA inhibitor increases the level of the NXPH1 protein and/or NXPH1 gene. A method of treating a disease or condition associated with aberrant levels of the PSD-95 protein and/or PSD-95 gene in a subject in need thereof, comprising a compound that inhibits miR-485 (miRNA inhibitor) to said subject, wherein said miRNA inhibitor increases levels of said PSD-95 protein and/or PSD-95 gene. A method of treating a disease or condition associated with aberrant levels of synaptophysin protein and/or synaptophysin gene in a subject in need thereof, comprising a compound that inhibits miR-485 (a miRNA inhibitor). The above method, comprising administering to the subject, wherein the miRNA inhibitor increases the level of the synaptophysin protein and/or synaptophysin gene. A method of treating a disease or condition associated with abnormal levels of caspase-3 protein and/or caspase-3 gene in a subject in need thereof, comprising a compound that inhibits miR-485 (miRNA inhibitor) to said subject, wherein said miRNA inhibitor reduces levels of said caspase-3 protein and/or caspase-3 gene. 46. The method of any one of claims 1-45, wherein the miRNA inhibitor inhibits miR-485-3p. 47. The method of claim 46, wherein said miR-485-3p comprises 5'-gucauacacggcucucucuccu-3' (SEQ ID NO: 1). 48. Any of claims 1-47, wherein said miRNA inhibitor comprises a nucleotide sequence comprising 5'-UGAUGA-3' (SEQ ID NO: 2), and said miRNA inhibitor is about 6 to about 30 nucleotides in length. 1. The method according to item 1. The miRNA inhibitor increases SIRT1 gene transcription and/or SIRT1 protein expression, increases CD36 gene transcription and/or CD36 protein expression, or increases PGC1 gene transcription and/or PGC1 protein expression. increase the transcription of the LRRK2 gene and/or the expression of the LRRK2 protein, increase the transcription of the NRG1 gene and/or the expression of the NRG1 protein, or increase the transcription of the STMN2 gene and/or the expression of the STMN2 protein or increases VLDLR gene transcription and/or VLDLR protein expression, increases NRXN1 gene transcription and/or NRXN1 protein expression, increases GRIA4 gene transcription and/or GRIA4 protein expression, Increase transcription of NXPH1 gene and/or expression of NXPH1 protein, increase transcription of PSD-95 gene and/or expression of PSD-95 protein, or increase transcription of synaptophysin gene and/or expression of synaptophysin protein or decreases caspase-3 gene transcription and/or caspase-3 protein expression, or any combination thereof. said miRNA inhibitor has at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides at said 5′ end of said nucleotide sequence , at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 50. Any one of claims 1-49, comprising 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides. the method of. said miRNA inhibitor has at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides at said 3′ end of said nucleotide sequence , at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 51. Any one of claims 1-50, comprising 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides. the method of. The miRNA inhibitor is 5'-UGUAUGA-3' (SEQ ID NO: 2), 5'-GUGUAUGA-3' (SEQ ID NO: 3), 5'-CGUGUAUGA-3' (SEQ ID NO: 4), 5'-CCGUGUAUGA- 3′ (SEQ ID NO: 5), 5′-GCCGGUGUAUGA-3′ (SEQ ID NO: 6), 5′-AGCCGUGUAUGA-3′ (SEQ ID NO: 7), 5′-GAGCCGUGAUGA-3′ (SEQ ID NO: 8), 5′- AGAGCCGUGUAUGA-3′ (SEQ ID NO: 9), 5′-GAGAGCGUGUAUGA-3′ (SEQ ID NO: 10), 5′-GGAGAGCCGUGUAUGA-3′ (SEQ ID NO: 11), 5′-AGGAGAGCCGUGUAUGA-3′ (SEQ ID NO: 12), 5 '-GAGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 13), 5'-AGAGGAGAGCCGGUGUAUGA-3' (SEQ ID NO: 14), 5'-GAGAGGAGAGCCGGUGUAUGA-3' (SEQ ID NO: 15), 5'-UGUAUGAC-3' (SEQ ID NO: 16) , 5′-GUGUAUGAC-3′ (SEQ ID NO: 17), 5′-CGUGUAUGAC-3′ (SEQ ID NO: 18), 5′-CCGUGUAUGAC-3′ (SEQ ID NO: 19), 5′-GCGUGUAUGAC-3′ (SEQ ID NO: 20), 5′-AGCCGUGUAUGAC-3′ (SEQ ID NO: 21), 5′-GAGCCGUGUAUGAC-3′ (SEQ ID NO: 22), 5′-AGAGCCGUGUAUUGAC-3′ (SEQ ID NO: 23), 5′-GAGAGCGUGUAUGAC-3′ ( SEQ ID NO: 24), 5′-GGAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 25), 5′-AGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 26), 5′-GAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO: 27), 5′-AGAGGAGAGCCGUGUAUGAC-3 5' (SEQ ID NO: 28), 5'-GAGAGGAGAGCGUGUAUGAC-3' (SEQ ID NO: 29), or AGAGAGGAGAGCGUGUAUGAC (SEQ ID NO: 30). the method of. The miRNA inhibitor is 5'-TGTATGA-3' (SEQ ID NO: 62), 5'-GTGTATGA-3' (SEQ ID NO: 63), 5'-CGTGTATGA-3' (SEQ ID NO: 64), 5'-CCGTGTATGA- 3′ (SEQ ID NO: 65), 5′-GCCGTGTATGA-3′ (SEQ ID NO: 66), 5′-AGCCGTGTATGA-3′ (SEQ ID NO: 67), 5′-GAGCCGTGTATGA-3′ (SEQ ID NO: 68), 5′- AGAGCCGTGTATGA-3' (SEQ ID NO: 69), 5'-GAGAGCCGTGTATGA-3' (SEQ ID NO: 70), 5'-GGAGAGCCGTGTATGA-3' (SEQ ID NO: 71), 5'-AGGAGAGCCGTGTATGA-3' (SEQ ID NO: 72), 5 '-GAGGAGAGCCGTGTATGA-3' (SEQ ID NO: 73), 5'-AGAGGAGAGCCGTGTATGA-3' (SEQ ID NO: 74), 5'-GAGAGGAGAGCCGTGTATGA-3' (SEQ ID NO: 75), 5'-TGTATGAC-3' (SEQ ID NO: 76) , 5′-GTGTATGAC-3′ (SEQ ID NO: 77), 5′-CGTGTATGAC-3′ (SEQ ID NO: 78), 5′-CCGTGTATGAC-3′ (SEQ ID NO: 79), 5′-GCCGTGTATGAC-3′ (SEQ ID NO: 79) 80), 5′-AGCCGTGTATGAC-3′ (SEQ ID NO: 81), 5′-GAGCCGTGTATGAC-3′ (SEQ ID NO: 82), 5′-AGAGCCGTGTATGAC-3′ (SEQ ID NO: 83), 5′-GAGAGCCGTGTATGAC-3′ ( SEQ ID NO: 84), 5′-GGAGAGCCGTGTATGAC-3′ (SEQ ID NO: 85), 5′-AGGAGAGCCGTGTATGAC-3′ (SEQ ID NO: 86), 5′-GAGGAGAGCCGTGTATGAC-3′ (SEQ ID NO: 87), 5′-AGAGGAGAGCCGTGTATGAC-3 ' (SEQ ID NO: 88), 5'-GAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 89), and 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 90). The method of any one of claims 49-51. the sequence of said miRNA inhibitor is 5′-AGAGAGGAGAGCCGGUAUUGAC-3′ (SEQ ID NO:30) or 5′-AGAGAGGAGAGCCGTGTATGAC-3′ (SEQ ID NO:90) with at least about 50%, at least about 55%, at least about 60%; 52. Any of claims 1-51 having at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity 1. The method according to item 1. 55. The method of claim 54, wherein the miRNA inhibitor has at least 90% similarity with 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:30) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:90). Claim 1, wherein said miRNA inhibitor comprises the nucleotide sequence 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:30) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:90) with one or two substitutions. 52. The method according to any one of the items 1 to 51. 52. The method of any one of claims 1-51, wherein the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:30) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:90). . 58. The method of claim 57, wherein said miRNA inhibitor comprises the nucleotide sequence 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:30). 59. The method of any one of claims 1-58, wherein said miR-485 inhibitor comprises at least one modified nucleotide. 60. The method of claim 59, wherein said at least one modified nucleotide is a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), an arabinonucleic acid (ABA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). . 61. The method of any one of claims 1-60, wherein said miRNA inhibitor comprises a backbone modification. 62. The method of claim 61, wherein the backbone modifications are phosphorodiamidite morpholino oligomers (PMO) and/or phosphorothioate (PS) modifications. 63. The method of any one of claims 1-62, wherein the miRNA inhibitor is delivered in a delivery agent. 64. The method of claim 63, wherein said delivery agent is a micelle, exosome, lipid nanoparticle, extracellular vesicle, or synthetic vesicle. 65. The method of any one of claims 1-64, wherein the miRNA inhibitor is delivered by a viral vector. 66. The method of claim 65, wherein said viral vector is AAV, adenovirus, retrovirus, or lentivirus. 67. The method of claim 66, wherein the viral vector is AAV with serotypes of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or any combination thereof. 68. The method of any one of claims 1-67, wherein the miRNA inhibitor is delivered by a delivery agent. 69. The method of claim 68, wherein said delivery agent comprises a lipidoid, liposome, lipoplex, lipid nanoparticle, polymeric compound, peptide, protein, cell, nanoparticle mimetic, nanotube, or conjugate. wherein the delivery agent is
[WP]-L1-[CC]-L2-[AM] (Formula I)
or [WP]-L1-[AM]-L2-[CC] (Formula II)
(In the formula,
WP is the water-soluble biopolymer moiety;
CC is a positively charged carrier moiety,
AM is the adjuvant moiety;
L1 and L2 are independently optional linkers, comprising a cationic carrier unit with
70. The method of claim 68 or 69, wherein the cationic carrier units form micelles when mixed with nucleic acids in an ionic ratio of about 1:1. 71. The method of claim 70, wherein said miRNA inhibitor interacts with said cationic carrier unit via an ionic bond. The water-soluble polymer is poly(alkylene glycol), poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharide) , poly(α-hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazoline (“POZ”), poly(N-acryloylmorpholine), or any combination thereof, or 71. The method according to 71. 73. The method of claims 70-72, wherein the water-soluble polymer comprises polyethylene glycol (“PEG”), polyglycerol, or poly(propylene glycol) (“PPG”). The water-soluble polymer has the formula:

74. The method of any one of claims 70-73, wherein n is 1-1000. wherein n is at least about 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about 121, at least about 122, at least about 123, at least about 124, at least about 125, at least about 126, at least about 127, at least about 128, at least about 129, at least about 130, at least about 131, at least about 132, at least about 133, 75. The method of claim 74, which is at least about 134, at least about 135, at least about 136, at least about 137, at least about 138, at least about 139, at least about 140, or at least about 141. The claim wherein n is from about 80 to about 90, from about 90 to about 100, from about 100 to about 110, from about 110 to about 120, from about 120 to about 130, from about 140 to about 150, from about 150 to about 160. 74. The method according to 74. 77. The method of any one of claims 70-76, wherein the water-soluble polymer is linear, branched, or dendritic. 78. The method of any one of claims 70-77, wherein the cationic carrier moiety comprises one or more basic amino acids. said cationic carrier moieties at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13 , at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38 , at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 79. The method of claim 78, comprising basic amino acids. 80. The method of claim 79, wherein said cationic carrier moiety comprises from about 30 to about 50 basic amino acids. 81. The method of any one of claims 79-80, wherein said basic amino acid comprises arginine, lysine, histidine, or any combination thereof. The method of any one of claims 70-81, wherein the cationic carrier moiety comprises about 40 lysine monomers. 83. The method of any one of claims 70-82, wherein said adjuvant moiety is capable of modulating an immune response, an inflammatory response, and/or a tissue microenvironment. 83. The method of any one of claims 70-82, wherein the adjuvant moiety comprises imidazole derivatives, amino acids, vitamins, or any combination thereof. The adjuvant moiety has the formula:

wherein each of G1 and G2 is H, an aromatic ring, or 1-10 alkyl, or G1 and G2 together form an aromatic ring, and n is 1-10. 84. The method according to 84. 85. The method of claim 84, wherein said adjuvant moiety comprises nitroimidazole. 85. The method of claim 84, wherein the adjuvant moiety comprises metronidazole, tinidazole, nimorazole, dimetridazole, pretomanide, ornidazole, megazole, azanidazole, benznidazole, or any combination thereof. 85. The method of any one of claims 70-84, wherein the adjuvant moiety comprises an amino acid. The adjuvant moiety has the formula:

(In the formula, Ar is

and each of Z1 and Z2 is H or OH. 85. The method of any one of claims 70-84, wherein the adjuvant moiety comprises a vitamin. 91. The method of claim 90, wherein said vitamin comprises a cyclic ring or cyclic heteroatom ring and a carboxyl or hydroxyl group. The vitamin has the formula:

92. The method of claim 90 or 91, wherein each of Y1 and Y2 is C, N, O, or S and n is 1 or 2. The vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D2, vitamin D3, vitamin E, vitamin M, vitamin H, and any of them. 93. The method of any one of claims 90-92, wherein the method is selected from the group consisting of combinations of 94. The method of any one of claims 90-93, wherein the vitamin is vitamin B3. said adjuvant moieties are at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10; at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 of vitamin B3. 96. The method of claim 95, wherein said adjuvant portion comprises about 10 vitamin B3. a water-soluble biopolymer moiety having about 120 to about 130 PEG units, a cationic carrier moiety comprising polylysine having about 30 to about 40 lysines, and about 5 to about 10 vitamin B3. 97. The method of any one of claims 90-96, comprising an adjuvant moiety comprising 98. The method of any one of claims 90-97, wherein the delivery agent is combined with the miRNA inhibitor to form micelles. 99. The method of claim 98, wherein said binding is covalent, non-covalent, or ionic. The cationic carrier unit and the miRNA inhibitor in the micelle are mixed in a solution such that the ionic ratio between the positive charge of the cationic carrier unit and the negative charge of the miRNA inhibitor is about 1:1. 100. The method of claim 98 or 99, wherein 101. The method of any one of claims 98-100, wherein said cationic carrier unit is capable of protecting said miRNA inhibitor from enzymatic degradation. 102. The method of any one of claims 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21-101, wherein said disease or condition comprises Alzheimer's disease. 2, 3, 5, 7, 9, 11, 13, wherein said disease or condition comprises an autism spectrum disorder, mental retardation, epileptic seizure, stroke, Parkinson's disease, spinal cord injury, or any combination thereof , 15, 17, 19, and 21-101. 104. The method of claim 103, wherein said disease or condition is Parkinson's disease. 64. The method of claim 63, wherein said delivery agent is a micelle. said micelles comprising: (i) about 100 to about 200 PEG units; (ii) about 30 to about 40 lysines each having an amine group; (iii) about 15 to about 15 each having a thiol group; 106. The method of claim 105, comprising about 20 lysines, and (iv) about 30 to about 40 lysines each bound to vitamin B3. The micelle comprises (i) about 120 to about 130 PEG units, (ii) about 32 lysines each having an amine group, (iii) about 16 lysines each having a thiol group, and ( 106. The method of claim 105, wherein iv) comprises about 32 lysines each bound to vitamin B3. 108. The method of claim 106 or 107, wherein a therapeutic moiety is further attached to said PEG unit. 109. The method of claim 108, wherein said targeting moiety is a LAT1 targeting ligand. 110. The method of claim 109, wherein said targeting moiety is phenylalanine.

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